A method for obtaining Schwann cell cultures from adult rabbit nerve based on "in vitro" pre-degeneration and neuregulin treatment.

Schwann cells (SCs) are basic elements for cell therapy and tissue engineering in the central and peripheral nervous system. Therefore, the development of a reliable method to obtain SC cultures is required. For possible therapeutic applications the cultures need to produce a sufficiently large number of SCs with a high level of purity in a relatively short period of time. To increase SC yield and purity we pre-degenerated pieces of 1-2 mm of adult rabbit sciatic nerves by incubating them for seven days in Dulbecco's Modified Eagle's Medium supplemented with 10% fetal bovine serum, penicillin/streptomycin and NRG1-ß1. Following pre-degeneration the nerve pieces were dissociated and then cultured for 6 or 15 days in the same culture medium. After 6 days of culture we obtained around 9.5x10³ cells/mg with approximately 94% SCs (S-100 positive) purity. After 15 days of culture the yield was about 80x10³ cells/mg and the purity was approximately 75%. Pre-degeneration and subsequent culture of small pieces of adult nerve with NRG1-ß1 supplemented medium increased the number of SCs and restricted the overgrowth of fibroblast-like cells.

Selective calcification of rat brain lesions caused by systemic administration of kainic acid.

Dystrophic calcification of previously damaged areas of nervous tissue occurs in a wide range of human diseases. The relationship between astroglial and microglial reactions and deposits of calcium salts was studied for up to five months in rats with a brain lesion produced by systemic administration of kainate. The morphology and atomic composition of the calcium salt deposits was also studied. Two types of lesions, sclerotic and liquefactive, were observed. In sclerotic lesions hyperplasia and hypertrophy of astrocytes partially substituted for the lost neurons, reaching a maximum in about twenty-five days after treatment. In liquefactive lesions, the astrocytic reaction occurred only around the liquefactive area. Microglial reaction was similar in both types of lesion and reached its highest expression in about twenty-five days. Calcium deposits were observed in the sclerotic but not in the liquefactive lesions. Clearly distinguishable granules of calcium salts were observed in sclerotic lesions under scanning electron microscopy after only five days post-injection. The size of calcified granules increased with time reaching 40 micro m or more in diameter at five months. The atomic composition of these deposits, studied by X-ray microanalysis, showed a time-dependent increase in calcium concentration. While there was no clear relationship between astroglial and microglial reactions and calcium salt deposits, the systemic injection of kainate produced progressively larger and more concentrated calcium deposits in sclerotic, but not in liquefactive lesions.

Neuronal nitric oxide synthase immunoreactivity in the guinea-pig liver: distribution and colocalization with neuropeptide Y and calcitonin gene-related peptide.

AIMS/BACKGROUND: The innervation pattern of the guinea-pig liver is similar to that of the human liver. However, many aspects of the distribution of the neuronal isoform of the enzyme nitric oxide synthase (nNOS) in the guinea-pig liver and its colocalization with neuropeptides remain to be elucidated. METHODS: The distribution of nNOS was studied in fixed guinea-pig liver by light microscopic immunohistochemistry. Confocal analysis was used to determine its colocalization with neuropeptide Y (NPY) or calcitonin gene-related peptide (CGRP). RESULTS: nNOS-immunoreactive (nNOS-IR) nerves were observed in relation to hilar and interlobar vessels and in Glisson's capsule. A few nNOS-IR ganglia were observed in the extrahepatic bile duct and close to the interlobar portal triads. In addition, nNOS-IR fibers were located in the interlobular portal triads and pervading the parenchyma. Moreover, nNOS-IR nerves were demonstrated for the first time in the larger central veins and in the hepatic vein. nNOS-NPY and nNOS-CGRP colocalizations were detected in the fibromuscular layer of the bile duct and periductal plexus, respectively. CONCLUSIONS: These results support the phylogenetic conservation of the nNOS-IR hepatic innervation and its possible contribution to the regulation of hepatic blood flow and certain hepatic functions.

Effects of unilateral deprivation in postnatal development of the olfactory bulb in an altricial rodent, the gerbil (Meriones unguiculatus).

To establish if olfactory bulb sensitivity to functional deprivation is related to the degree of development at birth, we studied the effects of surgical closure of one naris in the gerbil olfactory bulb development. The naris closure was performed at three different ages: at birth, P7 and P14 and maintained for 30 or 60 days. In coronal sections we measured total bulbar surface area and surface area of the different bulbar layers establishing an estimate multiple regression model for the percentage of surface area decrease in the deprived bulb related to non deprived one. The internal and external plexiform layers are the most sensitive layers to deprivation and age and duration of deprivation were factors in their mathematical models. The glomerular layer showed a surface reduction of about 25% without dependence either on age or duration. The deprived glomerular layer showed a much lower tyrosine hydroxylase-immunoreactivity and immunoreactive cell density than those in the non deprived one. However, differences in calbindin-immunoreactive and NADPH-diaphorase positive cell density between deprived and non deprived glomerular layer were not significant. Our results indicate that olfactory bulb sensitivity to functional deprivation is not related to the degree of precocity and changes in age and duration of deprivation cause different effects on the olfactory bulb layers.

Paravertebral muscles in experimental scoliosis: a light and electron microscopic study.

Experimental structural dextroconvex scoliosis was produced in rabbits by costotransversolisis with transversectomy and releasing of paravertebral muscles between TVII and TX on the right side. Two compensatory curves developed on the upper dorsal and lumbar levels. Biopsies of paravertebral muscles in experimental animals included, besides areas of normal tissue, a considerable derangement of the cell contractile apparatus with sarcoplasmic dilation and eventual cell disintegration and necrosis. Histological changes varied along levels, the convexity being more affected. The severity of changes and reduction in body weight and length were correlated with the degree of scoliosis. A selective atrophy of slow-twitch fibers was observed in experimental animals, especially at the level of the main curve, whereas fast-twitch fiber atrophy was more important caudally. Control animal biopsies always appeared normal. Our experimental model shows an overt participation of paravertebral muscles in the establishment of compensatory processes following scoliosis, although the role that paravertebral muscles play in the etiopathogenesis of human idiopathic scoliosis requires further investigation.

Prenatal development of the mammalian vomeronasal organ.

The vomeronasal organ (VNO) originates from the medial wall of the olfactory pit shortly after the middle of the embryonic period in mammals. The Anlage stage consists of a cellular bud that grows dorsally, caudally, and towards the midline leaving a groove. The following stage, Early Morphogenesis, includes the closure of the vomeronasal groove to form a parasagittal blind-ended tube in the nasal septum, which opens into the nasal and/or oral cavities. The lumen adopts a crescent shape while the epithelial lining differentiates into an increasingly wider epithelium on the concave side and a gradually thinner epithelium on the convex side. The former goes on to occupy a medial position and develops neuroblasts among supporting and undifferentiated cells, with supporting cell nuclei tending to align in the upper rows. The lateral "non-sensory" epithelium furrows, giving a kidney-shaped appearance to the VNO cross section. The next stage, Late Morphogenesis is extended up to a difference in thickness between both epithelia becomes similar to the adult, generally by birth. An increasing number of ciliary generation complexes, larger and more abundant microvilli, and an evident glycocalyx are observed in the neuroepithelium at the luminal surface, while enzymatic activities become more intense. The non-sensory epithelium appears quite mature save for its luminal surface, which is still devoid of cilia. Blood capillaries penetrate the most basal region of the neuroepithelium and vomeronasal glands are very few and immature. At birth, some neurons appear well developed to support certain functionality; however, persistence of architectural, histochemical, and ultrastructural signs of immaturity, suggests that full performance of the VNO does not occur in newborn mammals, but in prepubertal ages.

Brain lesions and water-maze learning deficits after systemic administration of kainic acid to adult rats.

The relationship between hippocampal damage and spatial learning deficiencies was studied in rats injected with kainic acid (10 mg/kg i.p.). A single injection was given either before or after the acquisition phase of the Morris water-maze task. In this acquisition phase, the animals were required to find a hidden underwater platform starting from four different points. The task was repeated twice a day for 10 days. In the retention phase after 10 days rest, the rats repeated the same task. The damage caused by the treatment occurred in several prosencephalic areas, including the piriform and enthorhinal cortices, the thalamus and the hippocampus. In the latter, greatest damage was seen in CA1 followed by CA3 while CA2 and the gyrus dentatus appeared almost unaffected. The behavioural results indicated that kainic acid impaired but did not preclude the acquisition of the water-maze task. During the retention phase, no significant differences in latencies were found between animals that were treated before and after acquisition, thus, indicating that pretraining does not play an important role in the recovery of these spatial abilities following hippocampal lesions.

Developmental stages of the vomeronasal organ in the rat: a light and electron microscopic study.

This paper describes the development of the rat vomeronasal organ from the stage of anlage until adulthood. Groups of four rats were sacrificed daily from prenatal day 13 (E13) until birth; at days 2, 4, 7, 10, 14 and 16 after birth; weekly from day P21 to P42 plus an additional group of adults. The vomeronasal organs were processed for light microscopy, including alcian blue-PAS and NADH-diaphorase reactions, and also for electron microscopy. For summarizing our results we propose the following developmental stages: 1. Anlage (E13). 2. Early morphogenesis (E14-16). 3. Late morphogenesis (E17 to birth). 4. Initiation of secretory activity (First postnatal week). 5. Cytoarchitectural maturity (2nd postnatal week). 6. Complete maturity (From 3rd postnatal week onwards). Our results on the maturation of the histological structure and the histochemical reactions, indicate that there may be some functional activity at birth but the development of the organ still continues during the first three postnatal weeks to acquire its full functional capability.

Postnatal development of the vomeronasal epithelium in the rat: an ultrastructural study.

Three basic types of cells are distinguished in the rat vomeronasal epithelium at birth: bipolar neurons, supporting cells, and basal cells. Neurons at this time include both immature and differentiated cells. By the end of the first postnatal week, all neurons show morphological signs of maturity in their cytoplasm, including abundant granular and smooth endoplasmic reticulum, neurotubules, dense lamellar bodies, apical centrioles, and tufts of microvilli. During the third week microvilli are more frequently encountered and appear to be longer and more branched. Supporting cells appear well-developed by the second day after birth. During the first ten days of life, supporting cells lose their centrioles and all of the complex associated with ciliary generation in the apical zone. Basal cells appear to be more numerous in newborns than in older animals. Protrusions projecting into the lumen are frequently observed in the epithelium of newborn animals, both on the dendrites of neurons and on supporting cells. After the third week, such protrusions are only observed in the transitional zone between the sensory and the non-sensory epithelia of the vomeronasal tubes. In this transitional zone, a fourth cell type showing apical protrusions with microvilli differentiates. Cytoplasm in this type resembles that of neighboring ciliated cells but has no cilia or centrioles. These transitional cells are considered to be cells in an intermediate state of differentiation, between that of the differentiated neurons and supporting cells of the sensory epithelium and that of the predominate ciliated cells of the non-sensory epithelium. The results suggest that by the end of the third week the vomeronasal epithelium is morphologically mature.

Quantitative histochemistry of cytochrome oxidase in rat brain.

A quantitative analysis of cytochrome oxidase (CO) activity in histochemically stained sections of rat brain was developed using tissue standards and computerized image processing. Standards of brain paste containing known amounts of CO were cryosectioned and stained under the same conditions as brain sections. The gray levels of the stain were converted to units of CO activity using a calibration curve derived from densitometric analysis of the standards. The technique yields reproducible quantitative values, has the superior anatomical resolution of histochemistry, and is compatible with autoradiography.

Histological development of the vomeronasal complex in the pre- and postnatal rat.

The vomeronasal complex of the rat was studied by means of different staining techniques and light microscopy between the 13th day of gestation and 42 days of postnatal life. The anlage of the vomeronasal organ in the 13-day-old embryo consisted of a cluster of cells proliferating from the olfactory placode towards the medial line. The vomeronasal organ was well developed by the end of gestation, showing the same ratio between receptor and receptor-free epithelium as in the adult animal. However, full development of the epithelia did not occur until the end of the second postnatal week. Glandular rudiments were observed within the vomeronasal area in 17-day-old fetuses, but the gland showed no sign of activity until the 7th day of postnatal life, when a slight PAS-positive reaction was observed in their cells' cytoplasm. This PAS-positive reaction intensified considerably during the second week and corresponded to the adult picture by the end of this time. The content of the vomeronasal glands contrasted with that of the glands flowing into the nasal fossa, which did not show any PAS-positive reaction but which stained positive for alcian blue. We also studied the evolution of the blood vessels such as the capsule of the complex. This capsule showed complete ossification by the beginning of the 3rd postnatal week, with the exception of certain regions which remained cartilaginous in the adult. Some capillaries were observed in the complex in 17-day-old fetuses, and by 20 days of gestation some of them could be observed penetrating the receptor epithelium. Subsequently, the vascularization of the complex became so profuse that one could consider the vomeronasal complex to consist of erectile tissue.