Biomedical subjects
E Marani
Publications and source records attributed to E Marani.
ELISA incubation times can be reduced by 2.45-GHz microwaves.
The ELISA (Enzyme Linked Immuno Sorbent Assay) technique is widely applied in the field of immunology. The use of this technique implies several incubation periods, often requiring more than one hour each. Microwave irradiation is known to be very useful in accelerating different kinds of processes. This knowledge has led to the present study in which it is proved that 2.45-GHz microwaves are able to reduce ELISA incubation times. This time reduction of 50% or more concerns each of the four incubation steps of the specific indirect ELISA which is used to determine the amount of antibodies in NF90-hybridoma cell culture supernatants. These incubation steps involve the coating of the microtiter plate, the incubation with BSA against non-specific binding, and the incubations with the first and second antibody. Combinations of more microwave-influenced steps in one ELISA save a considerable amount of time. When in addition air is blown through the incubation fluid during microwaving, the extinction values are almost the same as those of conventionally performed ELISAs. Thus, a good match of power setting and irradiation time results in a total incubation time reduction from 5.5 hours to 2 hours in this particular ELISA. Carefully monitoring the temperature of the incubation fluids during irradiation with the use of a fiberoptic thermometer appeared to be of crucial importance in the development of an optimal procedure.
Expression of vimentin and glial fibrillary acidic protein in the developing rat spinal cord: an immunocytochemical study of the spinal cord glial system.
The glial system in the developing rat spinal cord was studied using immunocytochemistry. Antibodies to vimentin and glial fibrillary acidic protein (GFAP) were used. At E11, vimentin was first found in the membrana limitans externa. In the matrix layer, short vimentin protrusions were found near the membrana limitans externa at E12. In addition, vimentin was scattered throughout the matrix layer, where it was also present as vimentin-positive tangles. Later in development, vimentin immunoreactivity was distributed in a distinct radial pattern in the matrix layer. During the first postnatal weeks, vimentin was replaced by GFAP which is therefore expressed in a similar radial pattern. This orderly structural organisation of vimentin and GFAP in the matrix layer could indicate the involvement of both proteins in morphogenetic processes such as neuron migration and cell organisation. In the mantle layer, a distinct radial vimentin immunoreactivity was replaced by GFAP immunoreactivity during the first 2 postnatal weeks. In addition, GFAP fibres appeared first, at E18, in the ventral mantle layer associated with the motor neuron columns. These glial fibres originated from a local source. In the dorsal mantle layer, GFAP-positive fibres were oriented tangentially, which is different from the overall radial arrangement. This expression pattern may be related to the ingrowth of primary afferents. In the ventral and dorsal raphe, a major vimentin expression was replaced by a minor presence of GFAP. Within the white matter, a vimentin-positive radial pattern was demonstrated which, after birth, was replaced by GFAP. This palisading pattern suggested an involvement of both proteins in the development and guidance of the ascending and descending spinal cord fibre systems. The general transition from the expression of vimentin to the expression of GFAP in the rat spinal cord takes place during the first 3 postnatal weeks.
The major importance of temperature data in publications concerning microwave techniques.
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The Microwave Newsletter incorporated in the European Journal of Morphology.
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Literature on microwaving in 1990.
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Innervation of annulus fibrosis in low back pain.
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Evidence for a rostrocaudal organization in dorsal root ganglia during development as demonstrated by intra-uterine WGA-HRP injections into the hindlimb of rat fetuses.
The development of the sensory innervation of the rat hindlimb was studied with special attention to the dorsal root ganglia and the lumbar plexus. Injections of wheat germ agglutinin-horseradish peroxidase were made into the hindlimb of 30 rat fetuses of gestational ages ranging from embryonic day 15-18. Additionally wheat germ agglutinin-horseradish peroxidase was applied to the sciatic nerves of 8 neonatal rats and 3 adults. The saphenous nerves of 2 neonatal rats were labeled. Injections of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) into the hindlimb of the fetuses result in completely and partially labeled dorsal root ganglia. Partial labeling always concerns the rostral or caudal part of a dorsal root ganglion. The associated dorsal roots of partially labeled dorsal root ganglia are also partially labeled in a corresponding rostrocaudal fashion. Reconstructions of the labeled nerves following injections into the hindlimb suggested that the somata of the sensory neurons of a particular nerve can be restricted to the rostral or caudal half of a dorsal root ganglion. For example: the rostral half of the fourth lumbar dorsal root ganglion belongs to the femoral nerve and its caudal half to the sciatic nerve. The results of application of wheat germ agglutinin-horseradish peroxidase to the central ends of the cut sciatic and saphenous nerves in neonatal rats confirmed these observations. So the rostrocaudal organization in the dorsal root ganglia stems from the distribution pattern of the peripheral nerves.
Atrophy of the hypothalamic lateral tuberal nucleus in Huntington's disease.
The hypothalamic lateral tuberal nucleus (NTL) was studied in the formalin-fixed brains of five patients with Huntington's disease (HD) and in five age- and sex-matched controls. With the Klüver-Barrera (luxol fast blue/cresyl violet) and hematoxylin and eosin stains the NTL was defined by its cytoarchitectonic characteristics. The nucleus was composed of one type of neuron and had about 60,000 cells. In HD, up to 90% neuronal loss was found in the NTL. The remaining neurons showed features of degeneration and there was astrocytosis. The estimated total number of glial cells in the NTL was reduced to 80% of the control values, which was exclusively accounted for by a reduction of 40% in the number of oligodendrocytes. The total number of astrocytes was unchanged. Grouping of astrocytes and the changes observed in glial fibrillary acidic protein immunocytochemistry suggested that astrocytic proliferation occurred.
The architecture of the magnocellular hypothalamus.
The distribution pattern of the magnocellular elements has been described using enzyme histochemical and classical staining procedures. The main amount of magnocellular neurons is found within nuclei, which, in addition are interconnected by strips of large neurons. These strips are organized as oblique lamellae traversing the hypothalamic area. The upper most lamella forms the dorsal hypothalamic border.
Somatotopic organization in the sensory innervation of the rat hindlimb during development, using half dorsal root ganglia as subsegmental units.
The aim of the present study was to determine the somatotopy of the sensory innervation of the rat hindlimb at the level of the dorsal root ganglia (DRGs) during the fetal and postnatal period. Injections of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) were made into the hindlimb of fetal and postnatal rats. These injections result in completely and partially labeled DRGs. Partial labeling always concerns the rostral or caudal half of a DRG. Using half DRGs as subsegmental units, a description is given of a somatotopic organization of the sensory innervation of the rat hindlimb during development.
Ventral root avulsions of the cat spinal cord at the brachial plexus level (cervical 7).
The ventral cervical 7th root was avulsed from the surface of the cat spinal cord, and studied using light microscopical stainings (Nissl, acetylcholinesterase and antibodies against neurofilament and glial fibrillary acidic protein) after different survival times. After two days the density of the neurofilament increased in the neurons in the ventral horn of the avulsed ventral root. Changes in the rough endoplasmatic reticulum (Nissl and acetylcholinesterase) started four days postoperatively, and were confirmed electron microscopically. The glial fibrillary acidic protein-positive structures surrounding the injured neurons in the avulsed ventral horn became more pronounced 30 days postoperatively. The number of neurons was definitely decreased 60 days after the avulsion. After the initial phase of the avulsion and before the distinct decrease in the number of neurons, the conditions for reimplantation of the avulsed ventral root and for the supposed regeneration can be expected to be more favourable for the neurons in the ventral horn.
Microwave-stimulated brain enzyme incubations are possible at the unphysiological condition of 50 degrees C.
Microwave-stimulated enzyme incubations for acetylcholinesterase, 5'-nucleotidase, alkaline phosphatase, lactate dehydrogenase, malate dehydrogenase, succinic dehydrogenase and isocitric dehydrogenase were studied, and compared with incubations in a waterbath. Temperature settings of 37 degrees C and 50 degrees C were used, and the incubation times were varied from 30 seconds to 30 minutes. The desired temperature of the incubation solution was reached in the microwave oven within 1 minute, whilst in the waterbath it took 10 to 25 minutes. The microscopic results for alkaline phosphatase and succinic dehydrogenase at a temperature setting of 50 degrees C were superior in the microwave method for incubation times less than 15 minutes. It is postulated that the increased reaction product of alkaline phosphatase and succinic dehydrogenase is due to a temperature effect, which has to be large enough to be of practical value. For the other enzymes studied, microwave-stimulated incubations were no better than the conventional incubations at corresponding temperatures. For 5'-nucleotidase there were aspecific lead deposits in the microwave method. All enzymes performed at the elevated, unphysiological temperature of 50 degrees C proved to have advantages, except for 5'-nucleotidase, whilst for malate dehydrogenase there was an aspecific reduction of the colour developer at this temperature.
The neurofilament architecture of the rat suprachiasmatic nucleus.
The neurofilament architecture within the suprachiasmatic nucleus of the rat was analyzed immunocytochemically using neurofilament monoclonal antibodies. The topographic distribution of neurofilament containing structures was restricted mainly to the ventral and caudal part of the suprachiasmatic nucleus, coinciding with the entrance area of the retino-suprachiasmatic fibres of this nucleus. Within the nucleus itself an axonal organization was present. The axons were grouped, forming clusters. These clusters existed of a core of myelinated axons surrounded by unmyelinated axons. The myelinated/unmyelinated axon ratio could reach 1:25. Within the nucleus the myelinated axons extended upwards to the middle part of the suprachiasmatic nucleus, where the fibers of the axon clusters fanned out.
Astrocyte-mediated induction of alkaline phosphatase activity in human umbilical cord vein endothelium: an in vitro model.
The blood-brain barrier, localized in the endothelium of the cerebral capillaries, is characterized by the existence of tight junctions, a low mitochondrial density, a low number of vesicles and a high activity of certain enzymes like alkaline phosphatase and gamma-glutamyl transpeptidase. Astroglial cells secrete a product that induces brain microvessel endothelial cells to differentiate into endothelial cells with blood-brain barrier properties. If rat astrocytes were grown together with human umbilical cord vein endothelial cells in a co-culture system in which there is no cellular contact between both cell types, alkaline phosphatase activity was induced in the endothelial cells after three days of co-culturing. If the endothelial cells were cultured in astrocyte conditioned medium, alkaline phosphatase activity was also induced, and preliminary results showed that formation of tight junctions occurred after five days. These observations support the hypothesis that astrocytes induce the differentiation of non-blood-brain barrier endothelial cells into endothelial cells with blood-brain barrier properties, in this study based on alkaline phosphatase-activity induction and induction of tight junction formation. These inductive processes are produced by a soluble factor released by the astrocytes.
The suprachiasmatic nucleus of the rat hypothalamus in culture: an anatomical and electrophysiological study.
This study shows that foetal neurons from the suprachiasmatic area, after dissociation and culture, contain in vitro the same characteristics as are found in the in vivo situation. The main peptidergic neurotransmitters present in the suprachiasmatic nucleus in vivo, vasoactive intestinal polypeptide (VIP) and vasopressin, are expressed in vitro while the cytoskeleton of these cells possesses phosphorylated neurofilaments. The exclusive uptake of Lucifer Yellow liposomes by neurons is also refound in suprachiasmatic cultures. The electrophysiological results are in agreement with those characteristics found in vitro and in vivo.
Acetylcholinesterase in the developing rat spinal cord: an enzyme histochemical study.
A descriptive enzyme histochemical study on the expression of acetylcholinesterase (AChE) in the developing rat spinal cord is presented. Between E11-E16, AChE was found to be associated with premitotic neurons of the ventral matrix layer, which indicated an involvement in the proliferation of the spinal cord motor neurons. From E12 on, AChE was abundantly present in the motor neurons of the ventral mantle layer, and in their fibres. This early expression suggested a function of AChE in the development of the motor neurons, rather than an active role in cholinergic transmission. The intermediolateral, the intermediomedial cell column and the region between these cell groups, were found to be positive for AChE. Cells of the adult intermediolateral cell column also expressed AChE. AChE, therefore, apparently plays a role in the development as well as in the functioning of the rat autonomic system. In the lateral funiculus, cells of the lateral spinal nucleus expressed AChE. After P8, AChE was expressed in the substantia gelatinosa. The enzyme may be associated with the fibre terminals of the primary afferents. AChE was found to be temporary expressed in the developing dorsal funiculus, which suggested a function of the enzyme in fibre growth and path-finding. At E12, AChE was located in the ventral aspect of the dorsal root ganglion. Later on, AChE positive cells were found throughout the ganglion.
Enzyme histochemistry of the spinal cord after experimental transection (Th 9) in the cat.
Subpial complete resection of a 10 mm segment of the spinal cord at Th 9 was performed in 9 adult cats. Topographic enzyme histochemical investigations of the terminal clubs were performed after different survival times after transection in 7 cats and three days after a subsequent one-week-delayed autologous sciatic grafting procedure in the remaining two cats. For acid phosphatase (ACP), the count of active terminal clubs was high (200 per m2) from 12 hours until day 3 after transection. Then the count of active terminal clubs decreased to a low level (20 per m2) and remained the same until day 14. Removal of necrotic tissue and subsequent grafting with autologous sciatic nerve did not change these findings. For succinate dehydrogenase (SDH), the numbers of terminal clubs showed the same pattern at a lower level. The SDH defined terminal clubs were smaller than the ACP ones. The length of the SDH positive area decreased after 7 days while the ACP positive area remained the same until day 14. The SDH active terminal clubs are overgrown by the ACP positive terminal clubs, after the 7th day. Considering that SDH is linked to constructive activity in mitochondria and ACP to destructive activity in lysosomes, this phenomenon might be responsible for the termination of the capacity of the spinal cord tissue to regenerate.