Markers for neuronal degeneration in organotypic slice cultures

This protocol describes ways of monitoring spontaneous or induced neuronal degeneration in organotypic brain slice cultures. Hippocampal cultures (4-week-old) are grown in normal serum-free control medium, or exposed to the neurotoxin trimethyltin (TMT) (0.5-100 microM) for 24 h or the excitotoxic glutamate agonist kainic acid (KA) (5-25 microM) for 48 h followed by 24 h or 48 h, respectively, in normal medium. Corticostriatal slice cultures (also 4-week-old) are exposed to KA (6-24 microM) for 48 h and normal medium for control. The resulting neurodegeneration is estimated by (a) propidium iodide (PI) uptake, (b) lactate dehydrogenase (LDH) efflux to the culture medium, (c) ordinary Nissl cell staining, (d) staining by the neurodegenerative marker Fluoro-Jade (FJ), (e) neuronal microtubule degeneration by immunohistochemical staining for microtubule-associated protein 2 (MAP2), and (f) Timm sulphide silver staining for heavy metal alterations. Both hippocampal and corticostriatal slice cultures show a dose- and time-dependent increase in PI uptake and LDH efflux after exposure to TMT and KA. The mean PI uptake and the LDH efflux into the medium correlate well for both types of cultures. Both TMT and KA exposed hippocampal cultures display in vivo patterns of differential neuronal vulnerability as evidenced by PI uptake, FJ staining and MAP2 immunostaining. Corticostriatal slice cultures exposed to a high dose of KA display extensive striatal and cortical degeneration in FJ staining as suggested by a high PI uptake. A change in Timm sulphide silver staining in deep central parts of some control cultures, corresponds to areas with loss of cells in cell staining, loss of MAP2 staining, PI uptake, and FJ staining. We conclude that organotypic brain slice cultures, in combination with appropriate markers in standardized protocols, represent feasible means for studies of excitotoxic and neurotoxic compounds.     

An in vitro blood-brain barrier model: cocultures between endothelial cells and organotypic brain slice cultures

This communication describes a novel in vitro blood-brain barrier (BBB) model: organotypic slice cultures from the central nervous system were overlaid on endothelial cell monolayers grown on permeable membranes. Morphological, electrophysiological, and microdialysis approaches were carried out to characterize and validate this model. After 10 days in coculture, morphological studies reveal the presence of tight junctions. Electrophysiological recordings of neuronal activity performed on organotypic cultures with or without an endothelial cell monolayer show that amplitude of evoked responses were comparable, indicating good viability of cocultures after 2 weeks. Perfusion of known BBB permeable or nonpermeable molecules was used to test the coculture tightness in conjunction with electrophysiological or microdialysis approaches: application of glutamate (Glu), which doesn't easily cross the BBB, triggers off rhythmic activity only in control cultures, whereas epileptogenic activity was observed in both control cultures and cocultures during perfusions with picrotoxin, a molecule that can diffuse through the BBB. Finally, the microdialysis technique was used to determine the permeability of molecules coming from the perfusion chamber: L-dopa, dopamine, and Glu were employed to assess the selective permeability of the coculture model. Thus, these results indicate that the in vitro model described possesses characteristics similar to those of the BBB in situ and that cocultures of organotypic slices and endothelial cell monolayers have potential as a powerful tool for studying biochemical mechanisms regulating BBB function and drug delivery to the central nervous system.     

Myelination in cerebellar slice cultures: development of a system amenable to biochemical analysis

Myelin deposition and maintenance are critical to proper function of the mammalian nervous system. Previous investigations of myelination in the central nervous system (CNS) were hampered by the lack of an in vitro system that can faithfully reproduce in vivo events yet is amenable to biochemical investigation. We have developed a procedure, based on organotypic cultures, which permits efficient preparation of large numbers of cerebellar slice cultures that can be easily manipulated. Cultures have been examined to document myelination biochemically (by incorporation of [35S]sulfate into sulfolipids), immunohistochemically (by labeling the myelin components myelin basic protein and galactocerebroside), and morphologically (by both light and electron microscopy). We tested the effects of biologically active peptides and antibodies on myelination in the thin slices. The results indicate that the cultures provide an in vitro system that can be used to examine specific cellular events that occur during CNS myelination.     

MR pancreatography: a useful tool for evaluating pancreatic disorders

Magnetic resonance (MR) pancreatography is being used with increasing frequency as a noninvasive alternative to diagnostic endoscopic retrograde pancreatography in the evaluation of the pancreatic duct and various pathologic conditions of the pancreas. This recently developed technique allows improved spatial resolution and permits imaging of the entire pancreatico-biliary tract during a single breath hold. MR pancreatography can help identify the course and drainage pattern of the pancreatic duct and is useful in diagnosing congenital anomalies such as pancreas divisum and annular pancreas without the risk of inducing pancreatitis. In some instances, MR pancreatography may demonstrate duct disruption and associated fluid collections resulting from trauma. In recurrent acute pancreatitis, MR pancreatography is useful in suggesting the cause of the disease; in chronic pancreatitis, it is useful in depicting ductal anatomy, detecting strictures or intraductal calculi prior to surgery, and detecting complications such as pseudocysts and fistulas. In addition, MR pancreatography performed in conjunction with abdominal MR imaging is useful in identifying pancreatic malignancies as well as in establishing resectability and preventing unnecessary preoperative stent placement.     

Neurotoxicity evoked by N-methyl-D-aspartate in the organotypic static slice cultures of rat cerebral cortices: effect of GABA(A) receptor activation

BACKGROUND: von Recklinghausen's neurofibromatosis is a hereditary disease that may affect any organ or system of the body primarily or secondarily, including the vascular system. Among the rare cerebrovascular abnormalities, the most common is stenosis or occlusion of the cerebral artery. Intracranial aneurysms are uncommon. CASE DESCRIPTION: A case of an intracranial cerebral aneurysm associated with von Recklinghausen's neurofibromatosis is reported. A 55-year-old woman presented with a history of intermittent headache for 2 months and right oculomotor nerve palsy for 1 month. Widespread cutaneous neurofibromas and angiomas were found over her trunk and limbs with prominent cafe-au-lait spots. X-ray showed that her left lung was compressed by a large mass in the left chest with rib defects and lateral spinal curvature. Right internal carotid angiography revealed a saccular aneurysm between C1 and C2. Craniotomy to clip the aneurysm could not be performed because the mass in her chest made intubation for general anaesthesia almost impossible. CONCLUSION: The clinical features of this case are discussed together with a review of 15 similar cases in the literature. There are different theories about this disorder. We agree that the malformations are derived not only from ectodermal, but also from mesodermal pathology. In terms of our case, we consider the progression of this disease to be slow.     

The psychological autopsy: a useful tool for determining proximate causation in suicide cases

This overview article examines the applications of the psychological autopsy method in determining proximate causation in suicide cases. The article reviews the history of the psychological autopsy and describes its procedure and how it has proved helpful in explicating proximate causation. The five standards currently used by the courts to determine proximate causation in suicide cases are described, as are a variety of applications of the psychological autopsy method, including its application to workers' compensation cases, product liability cases, and medical malpractice cases. In particular, issues of prediction and protection are addressed. Finally, there is a discussion of an application of the psychological autopsy to criminal cases. The article concludes with a discussion of the issues raised in the use of the psychological autopsy during expert testimony and the considerations that should be addressed by an expert witness contemplating the use of the psychological autopsy method.     

Magnetic resonance imaging: a useful tool for evaluation of bone prior to implant surgery

OBJECTIVE: To illustrate the potential for use of magnetic resonance imaging (MRI) to gain full sectional information before placement of osseo-integrated dental implants, with no patient exposure to ionising radiation. MATERIALS AND METHODS: Four typical cases are illustrated, the patients being imaged in a one tesla MRI scanner. The setting up of the sequences is explained, along with the use of an imaging/surgical template with gadolinium markers. RESULTS: MRI clearly shows full sectional detail of available bone for safe implant placement, and allows the delineation of cortical and cancellous bone to attain maximum implant length and stability. Vital structures and the floor of the maxillary sinus are clearly shown. CONCLUSIONS: MRI allows the surgeon to assess cases for suitability to place dental implants with confidence. The ability to scan directly at any desired plane, with no reformatting, and to relate this information to a surgical template gives predictable surgery. MRI is a sectional imaging modality giving information about the 3-dimensional relationship of the vital structures, without using ionising radiation. It therefore deserves consideration as an alternative to computed tomography. However, further work is indicated to investigate the relative technical merits of the two imaging modalities.     

Mood induction with older adults: A tool for investigating effects of depressed mood.

To validate the use of mood induction with older adults, 22 older participants receiving a depressed mood induction were compared with 20 controls and 17 older persons with high Center for Epidemiologic Studies-Depression Scale (CES-D) scores. The mood induction procedure, using self-referent statements and music, changed Depressive Adjective Checklist and CES-D scores in each of 2 sessions 1 week apart. Findings indicated that the use of mood induction procedures with older adults can provide experimental control of mood. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Novel staging tool for localized prostate cancer: a pilot study using genetic adaptive neural networks

PURPOSE: An estimated $1.5 billion is spent annually for direct medical expenses and an additional $2.5 billion for indirect costs for the management of prostate cancer. Today there are several procedures for staging prostate cancer, including lymph node dissection. Despite these procedures, the accuracy of predicting extracapsular disease remains low (range 37 to 63, mean 45%). Use of multiple staging procedures adds significantly to the costs of managing prostate cancer. Recently artificial intelligence based neural networks have become available for medical applications. Unlike traditional statistical methods, these networks do not assume linearity or homogeneity of variance and, thus, they are more accurate for clinical data. We applied this concept to staging localized prostate cancer and devised an algorithm that can be used for prostate cancer staging. MATERIALS AND METHODS: Our study comprised 1,200 men with clinically organ confined prostate cancer who underwent preoperative staging using serum prostate specific antigen, systematic biopsy and Gleason scoring before radical prostatectomy and lymphadenectomy. The performance of the neural network was validated for a subset of patients and network predictions were compared with actual pathological stage. Mean patient age was 62.9 years, mean serum prostate specific antigen 8.1 ng./ml. and mean biopsy Gleason 6. Of the patients 55% had organ confined disease, 27% positive margins, 8% seminal vesicle involvement and 7% lymph node disease. Of margin positive patients 30% also had seminal vesicle involvement, while of seminal vesicle positive patients 50% also had positive margins. RESULTS: The sensitivity of the network was 81 to 100%, and specificity was 72 to 75% for various predictions of margin, seminal vesicle and lymph node involvement. The negative predictive values tended to be relatively high for all 3 features (range 92 to 100%). The neural network missed only 8% of patients with margin positive disease, and 2% with lymph node and 0% with seminal vesicle involvement. CONCLUSIONS: Our study suggests that neural networks may be useful as an initial staging tool for detection of extracapsular extension in patients with clinically organ confined prostate cancer. These networks preclude unnecessary staging tests for 63% of patients with clinically organ confined prostate cancer.     

Neurons in the rat occipital cortex co-expressing the substance P-receptor and GABA: a comparison between in vivo and organotypic cultures

The morphology and the distribution of neurons expressing the NK1-receptor (NK1R) and the co-expression of gamma-aminobutyric acid (GABA) in these neurons were studied in the rat occipital cortex and in organotypic cultures (OTCs) derived from this structure. By employing immunohistochemistry, we demonstrate that the NK1R-expressing neurons are non-pyramidal neurons and co-express GABA. Some differences were noted between in vivo and OTCs. NK1R-expressing neurons in OTCs had larger somata and longer dendrites and the proportion stained with an anti-GABA-antibody (approximately 50%) was smaller than in vivo (90%). The preferential location of NK1R-expressing neurons in layers II/III and VI, seen in vivo is not present in OTCs where these neurons distribute rather homogeneously through layers II-VI. Our findings imply that in contrast to the cat and monkey, in the rat occipital cortex the effects of substance P are almost exclusively mediated via inhibitory interneurons.     

Creating and curating a pediatric radiology digital library to make the internet a useful reference tool for the radiologist

BACKGROUND AND OBJECTIVE: The endothelial cell count after phacoemulsification serves as a sensitive indicator for the level of corneal damage caused by different phacoemulsification techniques. PATIENTS AND METHODS: In a prospective and randomized study, the "Reversed Tip and Snip" technique and the "Divide and Conquer" technique were performed in groups of 30 patients each. The corneal endothelial cell count was measured preoperatively as well as 4 weeks and 3 months postoperatively. RESULTS: The endothelial cell count showed significant (P < .001) reduction by approximately 10% after the "Reversed Tip and Snip" technique and by approximately 15% (P < .001) after the "Divide and Conquer" technique. The latter produced a significantly (P < .001) greater cell loss. CONCLUSIONS: The "Reversed Tip and Snip" phacoemulsification technique produces less endothelial cell loss than the "Divide and Conquer" technique.     

Chemoenzymatic synthesis of iminocyclitol derivatives: a useful library strategy for the development of selective fucosyltransfer enzymes inhibitors

A chemoenzymatic strategy has been developed for the synthesis of libraries of iminocyclitol derivatives for the discovery of new and selective fucosidase inhibitors.     

External rhinoplasty: a useful approach for a young plastic surgeon

Among the frequently performed plastic surgery operations, rhinoplasty is the most difficult to obtain consistently good results. It is very challenging for young plastic surgeons to modify the external appearance of the nose and restore or maintain a good airway. The external approach has alleviated some of the problems due to understanding and learning of the rhinoplasty operation. Actually, open rhinoplasty allows better visualization of anatomical deformities and better manipulation of tissues allowing better integration of nasal anatomy and physiology. This paper reviews a personal series of 82 patients operated via an open approach between October 94 and October 97. During the same period, 24 patients were operated via an endonasal or percutaneous approach (osteotomies). Indications, advantages and disadvantages of the open approach are discussed. The various techniques used in this series are described and then critically analyzed. In open rhinoplasty, our present revision rate is 8.5%. In conclusion, the open approach seems to be useful for young rhinoplastic surgeons.     

Integrins: a role for adhesion molecules in olfactory memory

A gene required for short-term memory in Drosophila, Volado, encodes an alpha integrin and is preferentially expressed in the mushroom bodies of the adult brain. Adhesion molecules of this kind may play a role in olfactory memory by altering the strength of synaptic connections in an experience-dependent manner.     

Long-term cultures of human fetal liver cells: a three-dimensional experimental model for monitoring liver tissue development

BACKGROUND/AIMS: The present study describes an embryonic-fetal liver culture system which allows morphogenetic interactions consistent with the development of the hepatocellular function. METHODS: Intact livers from 8-12-week embryos were soaked in an extracellular matrix at 4 degrees C and gently dissociated without any enzymatic treatment. The resulting spherical hepatic units were cultured in a chemically defined serum-free medium and seeded into an extracellular matrix layer. Adherent three-dimensional tissue specimens were examined at various times by light and electron microscopy to evaluate the maintenance of hepatocyte morphology. RESULTS: The liver cells were viable for over 4 months; erythropoietic burst colonies were detected for longer than 6 weeks. Parallel detection of bile salt production in the medium by high performance liquid chromatography proved liver tissue functionality. Bile salt composition revealed predominance of taurine-conjugates rather than glycine. Maximum bile salt concentration (approximately 3 months) coincided with structural and ultrastructural observations indicating a marked decline in hematopoiesis, well-defined biliary canaliculi and formation of an organ-like structure. CONCLUSIONS: This three-dimensional culture system recapitulates fetal liver development with: (i) initial proliferation of both fetal erythropoietic and hepatic cells and (ii) subsequent shut-off of erythropoiesis and a shift to a more advanced stage of hepatocyte function, such as bile salt secretion.