Pores in synthetic nerve conduits are beneficial to regeneration.

Current opinion holds that pores in synthetic nerve guides facilitate nerve regeneration. Solid factual support for this opinion, however, is absent; most of the relevant studies assessed only morphological parameters and results have been contradictory. To evaluate the effect of pores, the rat sciatic nerve was either autografted or grafted with nonporous, macroporous (10-230 mum), and microporous (1-10 microm) biodegradable epsilon-caprolactone grafts. Twelve weeks later, the grafted nerves were resected, and the electrophysiological properties were determined in vitro. Subsequently midgraft-level sections were inspected, and peroneal nerve sections were evaluated morphometrically. Finally, the gastrocnemic and tibial muscle morphometrical properties were quantified. The microporous nerve graft performed much better than the nonporous and macroporous grafts with respect to most parameters: it was bridged by a free floating bundle that contained myelinated nerve fibers, there were more nerve fibers present distal to the graft, the electrophysiological response rate was higher, and the decrease in muscle cross-sectional area was markedly smaller. Hence, the present study demonstrates the beneficial effect of synthetic nerve guide pores on nerve regeneration, although with the caveat that not pores per se, but only small (1-10 microm) pores were effective.

Type grouping in skeletal muscles after experimental reinnervation: another explanation.

Type grouping signifies clustering of muscle fibres of the same metabolic type, and is a frequent finding in reinnervated muscles. To elucidate the mechanism behind it, the rat sciatic nerve was either autografted or grafted with hollow synthetic nerve grafts. Twelve weeks later the number and fibre area of the type I and type II muscle fibres in the gastrocnemic and anterior tibial muscles were determined after ATP-ase staining. The number and diameter of peroneal nerve fibres distal to the grafts were measured, and the number of Aalpha-nerve fibres was derived. Nearly all nerve and muscle morphometrical parameters changed equally in both experimental groups. However, type grouping occurred frequently only after autografting, whereas the number of nerve fibres and the number of Aalpha-nerve fibres increased in this group. Hence type grouping cannot be explained by increased intramuscular sprouting subsequent to a decrease in the number of innervating nerve fibres, as previously presumed. Regenerating axons branch along their course through the peripheral nerve. We propose that the probability of the occurrence of type grouping is related to the dispersion of sibling branches in the nerve. In the autograft, emerging branches are kept together by Schwann cell basal lamina scaffolds, in contrast to the hollow synthetic nerve grafts where the emerging branches become dispersed. Thus, in muscles reinnervated after autografting, the probability that nerve branches that arrive at a specific muscle territory are sibling branches is greater than after hollow tube grafting. Consequently, the probability that type grouping will occur is greater.

Adhesion and proliferation of human Schwann cells on adhesive coatings.

Attachment to and proliferation on the substrate are deemed important considerations when Schwann cells (SCs) are to be seeded in synthetic nerve grafts. Attachment is a prerequisite for the SCs to survive and fast proliferation will yield large numbers of SCs in a short time, which appears promising for stimulation of peripheral nerve regeneration. The aim of the present study was to compare the adhesion and proliferation of human Schwann cells (HSCs) on different substrates. The following were selected for their suitability as an internal coating of synthetic nerve grafts; the extracellular matrix proteins fibronectin, laminin and collagen type I and the poly-electrolytes poly(d-lysine) (PDL) and poly(ethylene-imine) (PEI). On all coatings, attachment of HSCs was satisfactory and comparable, indicating that this factor is not a major consideration in choosing a suitable coating. Proliferation was best on fibronectin, laminin and PDL, and worst on collagen type I and PEI. Since nerve regeneration is enhanced by laminin and/or fibronectin, these are preferred as coatings for synthetic nerve grafts seeded with SCs.

Intrinsic properties inhibit axonal outgrowth from neonatal rat spinal cord explant.

Lumbar spinal cord explants, harvested from neonatal rat pups aged between postnatal day 0 (P0) and P6, were cultured for a period of 48 hrs in the chemically defined medium R(12) on a poly-ethylene-imine (PEI) and on poly-D-lysin (PDL) coated surface. The outgrowth outside the explant was quantified. Lumbar explants from the same rat and embedded in a collagen matrix, and cortical explants from a P0 rat were used as controls. Statistical analysis demonstrated a clear relation between age-at-explantation and the number of neurites in the corona surrounding the explant. The number of outgrowing neurites decreased sharply with age-at-explantation. The average number of neurites per explant obeyed to the expression log (n) = -0.736x + 3.294 on PEI, and log (n) = -0.721x + 2.295 on PDL; x epsilon in [P0 - P6] (n, the number of neurites per explant; x, the age-at-explantation expressed in postnatal days). A similar observed age-related decrease of outgrowth has been described when culturing the lumbar explant inside a collagen matrix. The phenomenon appears to be an intrinsic property of the explant. We review growth inhibitory properties in different models and propose that the phenomenon occurs here at the interface explant-world.

Lumbar spinal cord explants from neonatal rat display age-related decrease of outgrowth in culture.

Lumbar spinal cord explants, harvested from neonatal rat pups aged between postnatal day 0 (P0) and P7, were cultured for a period of 48 h in the chemically defined medium R(12) [17] (Romijn, H.J., van-Huijen, F., Wolters, P.S., Neurosci Biobehav Rev, 8 (1984) 301-334), embedded in a collagen matrix. The outgrowth into the surrounding matrix was quantified. Age-matched cortical explants were used as controls. Despite adaptations of the culture protocol, outgrowth remained variable. Statistical analysis demonstrated a clear relation between the age of the explant (at the time of explantation) and the number of neurites in the corona surrounding the explant. The number of outgrowing neurites decreased sharply with age. The average number of neurites per explant obeyed to the expression log(N)= -0.652 A+17 (N: the number of neurites per explant; A: the age expressed in gestational days; A epsilon [G23-G30]; G23 signifying gestational day 23, or P0). The observed age-related decrease of outgrowth could not be explained by progressive myelination of the spinal cord white matter, nor by the absence of trophic support from muscle, but may be related to a progressive inability of the spinal neurites to interact with collagen.

Intrinsic properties of the developing motor cortex in the rat: in vitro axons from the medial somatomotor cortex grow faster than axons from the lateral somatomotor cortex.

The axons that originate in the medial somatomotor cortex of the rat depart, during development, after those from the lateral somatomotor cortex, yet they arrive in the cervical spinal cord first. Either the medially originating axons elongate faster, or the laterally originating ones pause along the descent pathway. To investigate the presence of an intrinsic difference of the axonal elongation velocity between the lateral and medial somatomotor cortical areas, we cultured explants taken from these areas for 2 days, and measured the length of the outgrowth. After 2 days the explants were surrounded by a radiate corona of axons of which the longest measured 1.95 mm. A significant difference was detected between the medial and lateral somatomotor cortical areas in vitro. Axons originating from explants taken from the medial somatomotor cortical area are, after 2 days in culture, on average 0.16 mm longer than those from the lateral somatomotor cortical area. Though the observed difference is not large enough to allow for the overtaking observed in vivo, it does indicate that intrinsic differences exist within the developing rat somatomotor cortex. This in turn indicates that intrinsic cortical traits not only influence regionalization and targeting behavior of cortical projection neurons, but also their axonal elongation speed.

The projections to the spinal cord of the rat during development: a timetable of descent.

In order to establish a timetable for the developmental descent of supraspinal descending projections in the rat, a retrograde neuronal tracer was injected into the spinal cord of rat fetuses and neonates both at different gestational ages and at different levels of the spinal cord. From the results of these experiments a position interval could be deduced for the leading descending fibers of each spinal-projecting nucleus at each age studied. The chronological series of position intervals of each supraspinal descending projection (the descent pattern) depicts the descent of its fiber front during development and allows for easy comparison between the various projections. According to these descent patterns the descent sequences of the various spinal-projecting nuclei were established. At E17 fibers from the lateral vestibular nucleus, the raphe magnus nucleus and the gigantocellular reticular nucleus were present in the lumbosacral spinal cord; their descent along the spinal cord thus occurs before this stage. At E18 fibers from the parafascicular prerubral nucleus, the interstitial nucleus of Cajal, the mesencephalic reticular nucleus, the caudal pontine reticular nucleus, the laterodorsal tegmental nucleus, the subcoerulean nucleus, the spinal vestibular nucleus, the interpolar spinal trigeminal nucleus, the raphe obscurus nucleus and the ventral medullary reticular nucleus arrived in the lumbosacral cord. At the same stage fibers from the oral and caudal spinal trigeminal nucleus reached their caudalmost extent in the spinal cord, respectively, the lower and upper thoracic cord. At E19 fibers from the oral pontine reticular nucleus, the parvocellular reticular nucleus, the ventral gigantocellular reticular nucleus and the ambiguous nucleus first appeared in the lumbosacral cord. At E20 fibers from Darkschewitsch's nucleus, the paralemniscal and parabrachial nuclei, cell group A5, the locus coeruleus, the gigantocellular reticular nucleus-alpha, the raphe pallidus nucleus, the paramedian reticular nucleus, and from the dorsal medullary reticular nucleus arrived in the lumbosacral cord. Last to arrive in the lumbar cord during the prenatal period, at E21, were fibers from the posterior commissural nucleus, the red nucleus, the Edinger-Westphal nucleus, the paragigantocellular reticular nucleus, the medial vestibular nucleus, Roller's nucleus, and the solitary nucleus. Fibers from the paraventricular hypothalamic nucleus and from the lateral hypothalamic area only arrived in the lumbosacral cord at P1, followed by fibers from the incertal nucleus at P4. A transient spinal projection from an unknown group of neurons located immediately lateral to and partly intermingled with the mesencephalic trigeminal nucleus arrived in the lumbosacral spinal cord at E18 and had disappeared at P1. This cell group, called Gr?, closely resembled the mesencephalic trigeminal nucleus (Me5). Both were teardrop shaped; an oblong mass of neurons at the caudal end with a long and thin trailing edge. The trailing edge of Gr?, however, curved dorsad towards the dorsal midline raphe of the caudal mesencephalon, while the trailing edge of Me5 curved rostrad, parallel to the sulcus limitans of the sylvian aquaduct. The neurons of Gr? are mainly round, but in the caudal part of the nucleus some horizontally oriented fusiform neurons were observed. All neurons of Gr? were tiny. These results confirm that the generation sequence of the source nuclei is not a prime determinant of descent sequence along the spinal cord. The distance between the source nucleus and the entrance to the target seems of influence only in the most extreme cases (diencephalic source nuclei and the cerebral cortex). Descent velocity of the fiber fronts is not equal between different sources, nor is the descent velocity of specific fiber fronts constant over time. (ABSTRACT TRUNCATED)

Topographical organization in the early postnatal corticopontine projection: a carbocyanine dye and 3-D computer reconstruction study in the rat.

We have explored basic rules guiding the early development of topographically organized projections, employing the rat corticopontine projection as a model system. Using anterograde in vivo tracing with 1,1',dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI), we studied the distribution of labelled fibers in the pontine nuclei in relation to cortical site of origin during the first postnatal week. Labelled corticopontine fibers enter the pontine nuclei in distinct, sharply defined zones. The putative terminal fibers typically occupy lamella-like subspaces. Related to changes in cortical site of origin, we describe mediolateral, internal to external, and caudorostral distribution gradients in the pontine nuclei. Fibers originating in the anterolateral cortex occupy an internal central core, while implantations at increasing distance from the anterolateral cortex produce 1) more externally located lamellae, and 2) a caudal to rostral shift in fiber location. Previous investigations have shown that pontocerebellar neurons migrate into the ventral pons in a temporal sequence (Altman and Bayer [1987] J. Comp. Neurol. 257:529). The earliest arriving neurons occupy the central core and later arriving neurons settle in more externally and rostrally located subspaces. We hypothesize that the earliest arriving corticopontine fibers grow into the then only available zone of pontocerebellar neurons (central core), attracted by a diffusible chemotropic cue. Later arriving fibers grow into correspondingly later and more externally and rostrally located contingents of pontocerebellar neurons. Thus, we propose that the topographical organization in the early postnatal corticopontine projection is determined by simple temporal and spatial gradients operative within source (cerebral cortex) and target region (pontine nuclei).

Simultaneous demonstration of CD15 and alkaline phosphatase activity in cryostat sections of rat fetuses: a detailed technical description for the developing brain.

CD15 and alkaline phosphatase are significant markers of the developing central nervous system. CD15 is known for its interaction in cell-cell contacts, while the presence of alkaline phosphatase is related to the formation of neuronal connections. This paper describes a combined immunocytochemical and enzyme histochemical technique to demonstrate both embryonic markers in the same cryostat section. The monoclonal antibody 3B9, which was used for the detection of CD15 recognizes the immunodominant carbohydrate structure alpha-1,3-Fucosyl-N-Acetyl-Lactosamine also known as X, LewisX, FAL or SSEA-1. This antigen dissolves and is easily rinsed out of the section. The procedure starts off with the detection of CD15. The results of the immunocytochemical procedure is a reaction product which is stable at a high pH, preventing loss of the immunocytochemical reaction product during the subsequent alkaline phosphatase detection. The other embryonic marker, alkaline phosphatase, can only be demonstrated enzymehistochemically if the enzyme is still active. The immunocytochemical localization procedure decided for, does not inactivate alkaline phosphatase totally. From the colorimetrical and histochemical alkaline phosphatase determinations it was concluded that the residual alkaline phosphatase activity detected with this technique, could be intensified by adding Mg(2+)-ions to both the colorimetrical and histochemical incubation media.

Retrograde labeling of retinal ganglion cells and brain neuronal subsets by [3H]-D-aspartate injection in the Syrian hamster hypothalamus.

The circadian pacemaker in the suprachiasmatic nuclei (SCN) is entrained to the environmental light-dark cycle via a direct retinal projection to the hypothalamus. This projection is thought to use glutamate or aspartate as neurotransmitter. [3H]-D-Aspartate was microinjected in the SCN and adjacent hypothalamic nuclei of Syrian hamsters. This neuronal tracer is selectively taken up by terminals of neurons that use glutamate or aspartate as neurotransmitter and retrogradely transported to their perikarya. With autoradiography labeled cells were visualized in the retinal ganglion cell layer. Labeled cells were also found in a subset of brain nuclei known to project to the injection area. Labeled cells were detected in the bed nucleus of the stria terminalis, paraventricular nucleus of the thalamus, lateral septal nucleus, and medial amygdaloid nucleus. No labeled cells were observed in the medial septal nucleus, intergeniculate leaflet, and ventral lateral geniculate nucleus, which are also known to project to the SCN. Our results indicate that glutamatergic/aspartatergic retinal ganglion cells project to the SCN and adjacent medial hypothalamic nuclei. Moreover, the SCN may receive glutamatergic/aspartatergic input from the brain neuronal subsets that were retrogradely labeled with [3H]-D-aspartate.

Prenatal descent of rubrospinal fibers through the spinal cord of the rat.

This study is the first description of the descent of rubrospinal fibers through the spinal cord of the rat fetus. Either horseradish peroxidase or wheat germ agglutinin-horseradish peroxidase conjugate was injected into the spinal cord, at different levels and at different gestational ages. At embryonic day 17 (E17) fibers from all subdivisions of the nucleus ruber (NR) started their descent towards the spinal cord. At E18 fibers from the ventrolateral NR reached the lower cervical spinal cord, and those from the caudal NR reached the lower thoracic spinal cord. At E19 fibers from the dorsomedial NR and from the parvicellular NR had just reached the cervical spinal cord, while fibers from the ventrolateral and caudal NR descended to lower thoracic levels. At E21 fibers from the dorsomedial NR reached the lower cervical spinal cord. Fibers from the ventrolateral and caudal NR completed their descent through the lumbosacral spinal cord during the first three postnatal days. During their descent the rubrospinal fibers were confined to the white matter of the spinal cord. The earliest descending fibers originated in the caudal NR. Fibers from the caudal part of each magnocellular subdivision of the NR descended before their rostral counterparts. Fibers from the dorsomedial NR only reached the cervical enlargement as the fibers from the ventrolateral NR descended through the cervical enlargement. The somatotopy of the adult rubrospinal projection reflects this sequence; the dorsomedial NR (dmNR) projects to the cervical spinal cord, and the ventrolateral NR (vlNR) projects to the lumbosacral spinal cord. In general, early descending fibers originated from neurons located caudally and ventrolaterally, while later descending fibers originated from neurons located progressively more rostrally and dorsomedially in the magnocellular NR.

Development of the spinal projections of the nucleus paraventricularis hypothalami of the rat: an intra-uterine WGA-HRP study.

The development of the spinal projections of the nucleus paraventricularis hypothalami (PVH) was studied by injecting wheatgerm-agglutinated horseradish peroxidase into the upper thoracic spinal cord of fetal rats at developmental ages ranging from embryonic day 16 (E16) to E21, and into the thoracic spinal cord of rat pups and adult rats. At E18 the first retrogradely labeled cells are seen in the PVH. These cells are located dorsally in the PVH, adjacent to the ventricular matrix. At E20 a more ventrally located group of retrogradely labeled cells appears. This ventral group joins caudally with the dorsal group of labeled cells. The dorsal group of labeled cells is closely packed, while the ventral group of labeled cells is intermingled with unlabeled cells. After E20 this spatial configuration remains essentially constant. Therefore descending projections from all spinal projecting cell groups in the PVH have reached lower cervical spinal cord levels at E20.

A zonal pattern of neuroglial cells during the development of the intermediate lobe of the rat pituitary.

In this paper the development of the intermediate lobe (IL) of the pituitary of the rat is described using alkaline phosphatase (AP) (EC 3.1.3.1.) enzyme histochemistry and stage-specific embryonic antigen 1 (SSEA-1) immunocytochemistry. SSEA-1 and AP are co-localized during late development and reveal the existence of two cytochemically different cell types within the IL, i.e. SSEA-1/AP-positive and SSEA-1/AP-negative cells. The SSEA-1/AP-positive cells are initially arranged along the hypophyseal lumen, in a number of longitudinally oriented zones. alpha-Melanocyte-stimulating hormone (alpha-MSH) immunoreactivity is expressed in the SSEA-1/AP-negative cells from E20 onwards. Eventually the SSEA-1/AP-positive cells develop into a layer of cells covering the luminal surface of the IL lobules. These cells represent the glio-epithelial or neuroglial cells of the IL.

The prenatal development of alkaline phosphatase activity in the hypothalamus of the rat.

The localization of alkaline phosphatase (E.C. 3.1.3.1.) positivity during prenatal development of the hypothalamus of the rat is described. At E12 all layers of the prosencephalon display alkaline phosphatase (AP) positivity. The AP positivity increases from dorsal to ventral. Within the hypothalamic area a second, rostro-ventral gradient exists from E14 onwards. At E18 both gradients have decreased. At E20 almost all AP positivity has disappeared from the hypothalamus, with the exception of some reaction product in the dorsal ventricular matrix of the hypothalamus. The significance of this pattern in relation to the differentiation of the hypothalamus and to the formation of hypothalamic connections is discussed. It is suggested that AP activity is related to the formation of connections.

A SEM study on the development of the ventricular surface morphology in the diencephalon of the rat.

The morphogenesis of the ventricular surface of the diencephalon of the rat was studied using scanning electron microscopy, cryostat serial sections and direct observations under a dissection microscope. Based on these observations a description is given of the neuromeres present within the prosencephalon and of the termination of the sulcus limitans. Two conclusions are reached. First, three neuromeres are present in the prosencephalon. Neuromere I consists of the telencephalon, the hypothalamic regions and the parencephalon anterius. Neuromere II is the parencephalon posterius, neuromere III the synencephalon. Second, the sulcus limitans terminates ventrally in the parencephalon posterius and does not continue towards the preoptic recess. No exact termination point of the sulcus limitans could be delineated.

The ontogeny of the spinocerebellar projection in the chicken. A study using WGA-HRP as a tracer.

This study investigates the ontogeny of the spinocerebellar projection and especially focuses on the relation between temporary longitudinal patterns in the cytoarchitecture of the developing cerebellum of the chicken and the emergence of longitudinal patterning in the spinocerebellar projection, WGA-HRP was injected into the spinal cord of chicken embryos at different stages of development. It was found that spinocerebellar fibers arrive in the cerebellar "anlage" before any longitudinal cytoarchitectonic pattern can be discerned. Thereafter the organization of the spinocerebellar projection seems to develop related to the two temporary cytoarchitectonic longitudinal patterns present in the chick cerebellum. In both stages the development of the spinocerebellar projection displays a rostro-caudal gradient.