Neuronal intermediate filament expression in rat dorsal root ganglia sensory neurons: an in vivo and in vitro study.

Qualitative and quantitative examination was performed to evaluate the expression of peripherin and 200 kDa neurofilament in the sensory compartment of the peripheral nervous system of the rat both in vivo and in a new in vitro model. Under physiological conditions, these two neuronal intermediate filaments show different expression patterns in sensory neurons. To have a more complete comprehension of the role of these intermediate filaments and to fill in the blanks left in previously reported literature, we demonstrate in vivo using a morphological approach that peripherin and 200 kDa neurofilament define two distinct subpopulations within the dorsal root ganglia sensory neurons. Moreover, peripherin is specifically expressed in unmyelinated fibers while 200 kDa neurofilament is expressed in myelinated fibers. Additionally, in vitro analysis of RNA taken from dorsal root ganglia explants suggested that 200 kDa neurofilament is downregulated and peripherin is transiently expressed throughout sensory fiber regrowth. In particular, both neuronal intermediate filaments are downregulated immediately after sensory fiber axotomy thus suggesting that neither peripherin nor 200 kDa neurofilament has a role in the first steps of fiber regrowth. However, the upregulation of peripherin a few days after the beginning of fiber regrowth in vitro suggests that low levels of peripherin may be require to carry on the sequence of events involved in the correct regeneration and direction of sensory fibers.

Lack of topographic specificity in nerve fiber regeneration of rat forelimb mixed nerves.

Multiple nerve repair by means of a Y-shaped nerve guide represents a good model for studying the specificity of peripheral nerve fiber regeneration. Here we have used it for investigating the specificity of axonal regeneration in mixed nerves of the rat forelimb model. The left median and ulnar nerves, in adult female rats, were transected and repaired with a 14-mm Y-shaped conduit. The proximal end of the Y-shaped conduit was sutured to the proximal stump of either the median nerve or the ulnar nerve. Ten months after surgery, rats were tested for functional recovery of each median and ulnar nerve. Quantitative morphology of regenerated myelinated nerve fibers was then carried out by the two-dimensional disector technique. Results showed that partial recovery of both median and ulnar nerve motor function was regained in all experimental groups. Performance in the grasping test was significantly lower when the ulnar nerve was used as the proximal stump. Ulnar test assessment showed no significant difference between the two Y-shaped repair groups. The number of regenerated nerve fibers was significantly higher in the median nerve irrespectively of the donor nerve, maintaining the same proportion of myelinated fibers between the two nerves (about 60% median and 40% ulnar). On the other hand, nerve fiber size and myelin thickness were significantly larger in both distal nerves when the median nerve was used as the proximal donor nerve stump. G-ratio and myelin thickness/axon diameter ratio returned to normal values in all experimental groups. These results demonstrate that combined Y-shaped-tubulization repair of median and ulnar nerves permits the functional recovery of both nerves, independently from the proximal donor nerve employed, and that tissue, and not topographic, specificity guides nerve fiber regeneration in major forelimb mixed nerves of rats.

HuC/D confocal imaging points to olfactory migratory cells as the first cell population that expresses a post-mitotic neuronal phenotype in the chick embryo.

In the present study, the expression of the HuC/D RNA-binding proteins, a marker of neurons that have left the mitotic cycle, in cells migrating from the olfactory neuroepithelium toward the telencephalon in the chick embryo was investigated by means of immunofluorescence and confocal laser microscopy. Results showed that this migratory cell population is early and massively labeled by the a-HuC/D antibody starting from the first olfactory pit stage. At this developmental stage, olfactory migratory cells appeared to be the only neuronal population that expressed the HuC/D antigens in the whole embryo. In following developmental stages, the great majority of migratory cells, the number of which increased progressively, continued to be heavily immunopositive for the a-HuC/D antibody while immunopositivity to this antibody begins to be detected in other regions of the nervous system. HuC/D immunopositivity persisted until stage 30 HH (about 6.5 days), the later developmental stage investigated in this study, when colocalization with GnRH was detected. Negativity to the anti-proliferating cell nuclear antigen (anti-PCNA) immunostaining, a marker of S-phase, showed that migratory olfactory cells have left the mitotic cycle. Altogether, these results suggest that we have identified the first population of post-mitotic neurons in the developing nervous system of the chick embryo.

Evidence of very early neuronal migration from the olfactory placode of the chick embryo.

Cell migration from the olfactory neuroepithelium is a very peculiar phenomenon in the development of the nervous system. In this paper, we provide evidence that, in the chick embryo, migration of cells from the olfactory neuroepithelium begins earlier than previously reported, namely at the same time as the first olfactory placode differentiation, that occurs several hours before the superficial ectodermal invagination that gives rise to the olfactory pit. Moreover, we provide evidence that very early migrating cells express the HuC/D RNA-binding protein antigen, a specific neuronal marker. These observations refocus our knowledge on the very first developmental stages of olfactory neuroepithelium.

Unscheduled DNA synthesis in rat adult myenteric neurons: an immunohistochemical study.

Unscheduled DNA synthesis refers to DNA synthesis not followed by cell division. Previous studies have suggested that this phenomenon may occur in neurons from peripheral myenteric ganglia in conditions of functional hyperstimulation. In order to verify these observations, we have carried on an immunohistochemical study on myenteric neurons from the hypertrophic intestinal loops upstream from a partial obstruction (an experimental condition that induces a relevant increase of the neuronal workload) after labelling with two different markers: the proliferating cell nuclear antigen (PCNA), that is specifically expressed in cell nuclei during the S-phase, and the protein gene product 9.5 (PGP 9.5), a specific marker of nerve cells. While no myenteric neuron immunopositive for the anti-PCNA antibody was found in the control intestine, in the hypertrophic myenteric ganglia some neurons were positive for PCNA. These results provide an unequivocal evidence on the existence of unscheduled DNA synthesis in myenteric neurons from the hypertrophic intestine.

Morphological and morphometrical changes in dorsal root ganglion neurons innervating the regenerated lizard tail.

The variations occurring in neurons from dorsal root ganglia that provide innervation to the regenerated tail of the lizard (vicarious ganglia) are analysed. Vicarious ganglion neurons, when compared to control ganglion neurons (i.e. ganglia from the same animal that were not involved in the reinnervation process), show a size increase of the soma (cell hypertrophy) which applies to all cell types and subtypes. No statistically significant differences in the relative percentage of neurofilament-poor (type D) and neurofilament-rich (type L) neurons were found between vicarious dorsal root ganglia compared to controls in all animals. On the contrary, within L neuron sub-types, a statistically significant increase in sub-type L2 (very rich in neurofilaments), and the appearance of sub-type L3 neuron which is not detectable in controls, were demonstrated in vicarious dorsal root ganglia. In spite of these variations in size and percentage distribution, no structural and ultrastructural differences of the various cell types and sub-types are detectable, except for the appearance of the sub-type L3 neurons. However, this neuron sub-type might not be considered specific of hypertrophy since the same morphological features have been observed, in normal conditions, in lizard dorsal root ganglia from cervical and lumbar spinal levels that provide innervation to limb plexuses.

Increase in the number and volume of myenteric neurons in the adult rat.

In response to enhanced functional activity, only the larger neurons of the myenteric plexus incorporate thymidine. Their number, too, is increased. This phenomenon is not accompanied by proliferation.

DNA content in neurons of Auerbach's plexus under experimental conditions in adult rats.

Some nerve cells of the Auerbach's myenteric plexus of the intestine of the adult rat, which hypertrophied following a surgically induced stenosis, began DNA synthesis unrelated to mitotic division. The cytophotometric analysis confirmed and quantified the amount of synthesis revealed by autoradiography with tritiated thymidine uptake. Numerous nerve cells show a DNA content exceeding the diploid level. Only a few of these show twice the diploid content. The significance of the DNA synthesis is discussed.

DNA neosynthesis in Auerbach plexus ganglia isolated from the rat hypertrophic gut: an electrophoretic analysis.

We analysed using electrophoresis the total genomic DNA extracted from isolated Auerbach plexus ganglia of the hypertrophic duodenum upstream from a partial experimental stenosis. Results indicated the presence of two extra-bands migrating below the high molecular weight DNA. suggesting that DNA amplification is the basic mechanism of the DNA neosynthesis previously observed in myenteric neurons.

Nucleo-plasmic index variability in dorsal root ganglion neurons of the lizard (Podarcis sicula) during neuronal hypertrophy.

The nucleo-plasmic index was investigated in lizard dorsal root ganglion neurons from different spinal levels (cervical, thoracic, lumbar and caudal) in normal conditions, as well as in caudal dorsal root ganglion (DRG) neurons innervating the regenerated lizard tail (caudal hypertrophic ganglia). Results showed no statistically significant difference in the distribution of nucleo-plasmic index values in DRGs from different spinal levels providing sensory innervation to peripheral territories of different size. On the contrary, in the caudal hypertrophic ganglia, a significant shift towards lower values of the nucleo-plasmic index was observed. This observation suggests that this 'old', and merely morphological, parameter could be regarded as a good and simple marker for the study of neuronal hypertrophy considered as a dynamic response of neurons to variations of the innervation territory.

Electrophoretic analysis of neuronal genomic DNA from hypertrophic spinal ganglia during lizard tail regeneration.

Cytoplasmic and nuclear hypertrophy in neurons from the last 3 pairs of sensory ganglia left in situ cranially to the plane of amputation occurs during lizard tail regeneration. Cytophotometry after Feulgen staining demonstrated the presence of some neurons, from hypertrophic ganglia, whose quantity of DNA exceeded the diploid level (hyperdiploid neurons). In the present work agarose gel electrophoresis of total genomic DNA extracted from hypertrophic ganglia showed one or two bands migrating below the high molecular weight DNA, pointing to a selective amplification of discrete DNA segments.

Morphological analysis of peripheral nerve regenerated by means of vein grafts filled with fresh skeletal muscle.

Clinical data have shown that a vein segment filled with fresh skeletal muscle can be considered a good autologous grafting conduit for the repair of peripheral nerve lesions. In this study, the long-term morphological organization of rat sciatic nerve fibers regenerated along a muscle-vein-combined graft conduit is further analysed by light and electron microscopy. Regenerated nerve fibers were organized into fascicles of various sizes that were clearly delimited by perineurial-like shells made by long and thin cytoplasmic processes of perineurial-like bipolar cells and by densely packed collagen fibrils. Grafted skeletal muscle fibers were still detectable among nerve fiber fascicles. However, in spite of the persistence of skeletal muscle along the graft, regenerated nerve fibers showed a good morphological pattern of regeneration, providing further evidence that the muscle-vein-combined grafting technique represents an effective surgical alternative to the classical fresh nerve autograft for the repair of peripheral nerve defects.

Methodological issues in size estimation of myelinated nerve fibers in peripheral nerves.

Size estimation of myelinated nerve fibers in peripheral nerves is a very common task in neuromorphology and different dedicated morpho-quantitative procedures have been devised and used to date. Unfortunately, many reports on experimental nerve studies lack comprehensive information on the procedures that have been designed and applied for myelinated fiber size estimation. This paper addresses the issue in the light of the recent advances in quantitative morphology that have recognized the concept of unbiased estimates as the key methodological issue to be addressed in morpho-quantitative studies. The potential foundations of bias at various study levels are analysed together with indications on how to cope with them. In addition, the issue of the precision of size estimates is addressed and the various geometrical parameters that can be selected for myelinated nerve fiber size assessment are outlined. Taken together, information provided in this paper is expected to help investigators conduct an appropriate preliminary study design phase, the key step for setting up the most adequate morpho-quantitative procedure for any given research goal.

Morphometrical analysis of types and sub-types of neurons in dorsal root ganglia of the lizard Podarcis sicula.

In the present study, we conducted a morphometrical analysis of the different types and sub-types of lizard DRG neurons at various spinal levels. This analysis demonstrated significant differences in size distribution among the various neuron types and sub-types, as well as a significant shift to greater values in neurons from the dorsal root ganglia at the cervical and the lumbar spinal levels. The results are critically evaluated in relation to methodological issues, and the implications of these findings are discussed.

Types and sub-types of neurons in dorsal root ganglia of the lizard Podarcis sicula: a light and electron microscope study.

A morphological analysis of types and sub-types of neurons from dorsal root ganglia at different spinal levels was carried out by combined light and electron microscopy in Podarcis sicula. Two neuron types were recognized: small dark cells (type D) and large light cells (type L). Type L cells were further sub-divided into three sub-types (L1, L2, L3) on the basis of entity and distribution of neurofilaments. Percentage distribution of neuron types did not vary in relation to the spinal level. On the contrary, differences were found in the percentage of the three type L neuron sub-types; a higher percentage of cells very rich in neurofilaments (L2, L3) was found in dorsal root ganglia from the cervical and the lumbar spinal levels. Results are discussed in relation to the spinal-level-related extent of innervation territory of dorsal root ganglia.

Functional and morphological assessment of a standardized crush injury of the rat median nerve.

The availability of effective experimental models for investigating nerve regeneration and designing new strategies for promoting this unique repair process is important. The aim of this study was to standardize a rat median nerve crush injury model using a non-serrated clamp exerting a compression force of 17.02 MPa for a duration of 30s. Results showed that functional recovery, evaluated by grasping test, was already detectable at day-12 and progressively increased until day-28 after which animal performance plateaued until the end of testing (day-42), reaching a range of 75-80% of pre-operative values. Morphological analysis on the median nerve segments, distal to the crush lesion, which were withdrawn at the end of the experiment showed that regenerated nerve fibers are significantly more numerous and densely packed; they are also smaller and have a thinner myelin sheath compared to controls. Together, these results provide a baseline characterization of the crush median nerve injury experimental model for its employment in the investigation of nerve regeneration research, especially when a reproducible regeneration process is required, such as for the study of biological mechanisms of peripheral nerve fiber regeneration or development of new therapeutic agents for promoting posttraumatic nerve repair.

The use of brainstorming for teaching human anatomy.

Interactive teaching techniques have been used mainly in clinical teaching, with little attention given to their use in basic science teaching. With the aim of partially filling this gap, this study outlines an interactive approach to teaching anatomy based on the use of "brainstorming." The results of the students' critique of the teaching techniques are also included. Seventy-five students from the first-year nursing curriculum were tested by a structured questionnaire after three brainstorming sessions. The overall response to these sessions was very positive, indicating that students perceived this interactive technique as both interesting and useful. Furthermore, this approach may provide a useful strategy when learning the clinical courses of the upcoming academic years.

Smooth muscle cell hypertrophy and hyperplasia in the partially obstructed gut of the rat: a quantitative evaluation.

Partial surgical stenosis of the gut induces smooth muscle cell hypertrophy and hyperplasia in the loops upstream from the obstruction in a few days. In the present study we report a quantitative evaluation of these phenomena in the circular smooth muscle layer of the small intestine of the rat 7 days after a subtotal stenosis. In the loops upstream from the obstruction, lumen diameter and muscle wall thickness were found to be increased in comparison with downstream tracts. Morphometrical analysis showed that cross-sectional profile areas of smooth muscle cells, within the circular layer of upstream loops, significantly increased in size. Moreover, smooth muscle underwent a marked cell hyperplasia; in fact, estimates of the number of smooth muscle cell nuclear profiles turned out to be from 2.0 to 3.8 times greater in upstream loops than in downstream loops. The relation between the degree of lumen dilatation and the degree of the increase of the circular muscle layer thickness and of hypertrophic and hyperplastic response is discussed.