A morphometric study of optic axons regenerated in a sciatic nerve graft of adult rats

PURPOSE: In the present study we have morphometrically examined a regeneration model in which axons normally residing in CNS have regrown and are interacting with Schwann cells from the PNS. This study will not only provide morphometric data on regenerated optic fibers but also shed light on possible factors in determining the fiber morphometry. METHODS: The optic nerves of rats aged 6 weeks were cut intra-orbitally and replaced with a autologous sciatic nerve. After a survival period of 9 months, the graft or "regenerated" nerves containing the regenerated optic axons and Schwann cells were processed for morphometric measurements. RESULTS: The mean myelinated axon diameter of regenerated nerve (1.8 +/- 0.2 micro m) was significantly (P < 0.05) greater than that of the optic nerve (0.9 +/- 0.03 micro m). However, unmyelinated regenerated optic axons had a smaller mean axon diameter (0.49 +/- 0.04 micro m) than normal myelinated optic axons. This may suggest that myelinating glial cells exert an influence on axon caliber and Schwann cells seem to have greater effect than oligodendrocytes. The mean g-ratio showing the relative myelin sheath thickness was found to be the highest in the optic nerve (0.78 +/- 0.003), least in the sciatic nerve (0.6 +/- 0.009) and intermediate in the regenerated nerve (0.68 +/- 0.01). The results indicated that Schwann cells myelinating the regenerated optic axons have produced a thinner myelin sheath. Intra-axonally, no significant difference was detected in the number of axonal microtubules and neurofilaments between the regenerated and optic nerves. Therefore the disposition of microtubules and neurofilaments into axon may be intrinsically determined. CONCLUSIONS: In this study, we have identified some of the extrinsic and intrinsic factors in determining the fiber morphometry of the regen-erated nerve. The axon-size and myelination by glial cells were determined through the external axon-glial interactions, whereas the number of axonal microtubules and neurofilaments were intrinsically determined.     

A contribution to the electron microscopic morphometric analysis of peripheral nerve.

Several aspects of data collection and analyses of peripheral nerve experiments employing light and electron microscopic morphometric techniques have not been adequately discussed in the literature. From statistical tests performed on nerve data, it was found that light compared with electron microscopic morphometry underestimates the number of small fibers. An optimum sampling strategy must take into account a potential bias toward small fibers introduced by measuring fibers from electronmicrographs. It must also take into account a potential bias introduced by the non-random distribution of nerve fibers of different sizes in nerves. These biases are offset by sampling a large enough number of fibers from large enough area electron micrographs. A method is presented for analysing periopheral nerve data using the nested analysis of variance. This requires first dividing the usual bimodal nerve fiber distribution into component normally distributed parts. The number of fibers in the two portions of a bimodal distribution must be considered in data analysis. Knowledge of the variances of parameters to be studied in any particular nerve is necessary for optimum sampling strategies.     

The postnatal development of the optic nerve in hamsters: an electron microscopic study

The postnatal development of the optic nerve in the golden hamster has been examined using the electron microscope. The number of the optic fibres present in the optic nerve showed an initial increase during the first day after birth but it declined afterwards and tapered off after Day 16. At its peak on postnatal Day 1, there was an average of 314,629 axons in the optic nerve but when the animal reached adulthood only 109,587 fibres remained amounting to about 65% loss of the total fibre population. The period of axon loss coincided with the appearance of large patches of degenerated profiles in the optic nerve. The occurrence of the optic fibre loss has been implied to correlate with the time when the retinal projections were undergoing a dynamic reorganization at the target sites during the process of establishing the adult patterns of retinofugal connections.     

Early uncrossed component of the developing optic nerve with a short extracerebral course: a light and electron microscopic study of fetal ferrets

During the study of the developing optic nerve described in the preceding paper (Guillery and Walsh, '87), small bundles of nerve fibers were seen passing between the optic nerve and the ipsilateral hypothalamus of 24-to 27-day-old prenatal ferrets. The bundles appear before any other fiber groups of the retinofugal pathway and are identifiable while the main portions of the retinofugal system are growing into the optic tracts. The bundles, made up of 50 or more axons, leave the optic nerve, emerge through the otherwise continuous layer of subpial glia and through the basal lamina of the nerve, run a short, naked, extracerebral course among collagen fibers and presumed fibroblasts, and then re-enter the central nervous system, passing rostrally and dorsally to the superficial parts of the ipsilateral hypothalamus away from the region of the chiasm. These fibers represent the earliest link between the optic nerve and the brain, but their course is not followed by the majority of retinofugal fibers developing later, which pass toward one or the other optic tract.     

A quantitative study of developing axons and glia following altered gliogenesis in rat optic nerve

Axonal and glial cell development within rat optic nerve in which gliogenesis was altered by systemic injection of 5-azacytidine (5-AZ) was examined by quantitative electron microscopy. In neonatal (0-2 days) rat optic nerves, all fibers are premyelinated, and they exhibit a fairly uniform diameter (approximately 0.22 micron). These fibers occupy approximately 55% of the optic nerve volume. At this early age, glia within the optic nerve consist only of cells of astrocytic lineage and progenitor cells. These glia occupy approximately 28% of the optic nerve volume, and there are approximately 80 glial cells/optic nerve cross section. In 14-day-old normal optic nerves, myelinated and ensheathed fibers comprise approximately 17% and 9%, respectively, of the total number of axons. Mean axonal diameter of myelinated fibers is approximately 0.75 micron, while mean diameter for ensheathed axons is approximately 0.50 micron. By volume, these fibers occupy approximately 25% of the nerve, which is similar to the volume occupied by premyelinated axons in these nerves. At 14 days of age, there are approximately 300 glial cells/optic nerve transverse section, and these glia occupy approximately 37% of the volume in normal optic nerve. Oligodendroglia represent approximately 40% of total glial cells present, while astroglia and progenitor cell each comprise approximately 30% of the cells. In optic nerves from 14-day-old rats treated with 5-AZ, few myelinated fibers are present and the number of oligodendroglia is markedly reduced. Axonal diameter of premyelinated fibers is similar to that of age-matched controls. Myelinated and ensheathed fibers comprise approximately 2% of the total fibers present in 5-AZ-treated optic nerves, with the remaining fibers being premyelinated. The few myelinated and ensheathed fibers present in 5-AZ-treated optic nerves display similar axonal diameters to corresponding fibers from age-matched control tissue. Glial cells occupy approximately 40% of the nerve volume, and there are approximately 200 glia/nerve cross section in 5-AZ-treated rats. Astroglia comprise approximately 63% of the total glial cells, while approximately 12% of the cells are oligodendroglia. These results demonstrate that 5-AZ is a potent inhibitor of oligodendrogliogenesis, with a concomitant marked reduction in the number of myelinated fibers.     

A morphometric analysis of extracellular space in the developing spinal cord of the chick embryo

The developmental changes in the amount and distribution of the expanded extracellular space (ECS) (i.e. wider than 100 nm) were analyzed in the cervical spinal cord of chick embryos between stage 9 and 29, using electron micrograph montages, which cover one half of the cross-sectional area of the cord. The percentage of the ECS expansion to the whole cross-sectional area of the cord was 11.0% at stage 9, 7.7% at stage 11, 7.8% at stage 15, and 9.7% at stage 17. It decreased markedly to 3.0% at stage 22 and 1.3% at stage 29. The highest percentage at stage 9 may reflect the dynamic structural changes associated with neural groove closure which takes place around this time. The marked decrease after stage 22 is associated with the rapid overall growth of the cord. Until stage 19, the ECS expansions were mostly elongated and arranged radially with respect to the central canal. The ECS became scarce and arranged randomly thereafter. Throughout the stages examined, especially between stages 17 and 19, percentage was higher in the outer half of the cord than in the inner half. The outer glial limiting membrane was not established by stage 29. Between stages 17 and 22, the percentage was higher in the dorsal region than in the ventral region. This appears to be associated with the regional difference in neuronal maturation. The first blood vessels penetrated the ventromedial portion of the cord around stage 22, where the ECS expansions were relatively scarce. The successive rapid decrease in the amount of ECS expansions can be correlated to the development of vascularization.     

Growth pattern of axons in the optic nerve of chick during myelogenesis

The purpose of this experiment was to study the diameter of axons at the time of the initiation of myelin and the pattern of growth of axons in the optic nerve of the chick. Embryos between 15 and 20 days and chicks 3, 5, 22 and 60 days of age were studied on the electron microscopic level. Based on axon diameter a unimodal distribution of unmyelinated axons is present through day 20 of incubation with a mean of approximately 0.35 micrometer. This population is represented through 22 days of age but from day 3 on, a second distinct population of unmyelinated axons is present which has a mean diameter that is approximately twice that of the smaller unmyelinated axons. All axons do not increase simultaneously in diameter but once growth starts, the unmyelinated axons apparently double in diameter at a relatively rapid rate prior to myelination. On incubation day 17 less than 1% of the axons in the optic nerve is myelimated. The number of axons in this group and their diameter (mean approximately 1.2 micrometer) remain relatively constant through day 3 but from days 5 through 22, two distinct populations of myelinated axons are present. By day 60, three distinct distributions of myelinated axons are present with mean diameters of 0.51 micrometer, 1.76 micrometer, and 3.90 micrometer. These populations represent approximately 20%, 67%, and 13% respectively of the total fiber population. As age increases the diameter of some myelinated axons is as small as or smaller than the unmyelinated axons at an earlier period in development. This suggests that factors other than axon diameter might be involved in the start of myelination. It appears that the increase in axon diameter does not occur in a continuous manner but in a saltatory manner from one size to another.     

The course of axons of retinal ganglion cells within the optic nerve and tract of the chick (Gallus gallus)

Small laser lesions placed in the posthatch chicken retina resulted in axotomy and then death of all ganglion cells located in a sector peripheral to the primary damage. With the use of silver techniques, the patterns of degenerating retinal fibers in the optic nerve, chiasm, and optic tract were examined. In the proximal part of the optic nerve, radial retinal lesions resulted in a sheet of degenerating axons along the rostrocaudal extent of the nerve. The position of degenerating axons was related to the site of their entry in the optic nerve head with an overlapping distribution of degenerating fibers entering the optic nerve head from equivalent points from the temporal and nasal sides. In the optic chiasm, the distribution of fibers was similar to that seen in the proximal part of the optic nerve. In the optic tract there was a similar mixing of fibers from opposite sides of the retina. The ventral, nasal and temporal retinal fibers lay in the superficial part of the tract whereas the fibers from the nasal and temporal dorsal retina ran in the deeper, medial aspect of the tract. The central-to-peripheral axes of the retina were mapped along the rostrocaudal axis of the tract. As the tract approached the tectum degenerating fibers from single retinal lesions did not always remain together. In the case of a lesion in the ventral nasal retina, degenerating fibers split into two bundles located at opposite ends of the tract only to reunite at their terminal regional at the caudal pole of the tectum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Some aspects of synaptogenesis in the spinal cord of the chick embryo: a quantitative electron microscopic study.

We have quantitatively examined the development of synapses in the ventral part of the lumbar spinal cord of the chick from embryonic day 4 until adulthood. The first synapses occur on day 4 and are of the axo-dendritic type; they are invariably located adjacent to the border between the intermediate and marginal zones. Initially there are more synapses in the presumptive white matter than in the motoneuron neuropil, but this trend is later reversed; however, we found numerous axo-dendritic synapses throughout much of the ventrolateral white matter even in the adult stage. The first axo-dendritic synapses always contain spherical synaptic vesicles and have symmetric membrane specilizations. By day 7 a few of these synapses were found to have mixed populations of spherical and flattened vesicles and asymmetric membrane specilizations. After hatching there are still considerably more axo-dendritic synapses with symmetric membrane specializations. Axo-somatic synapses were first found on embryonic day 6 and were typically located on motoneurons lying adjacent to the marginal zone. These axo-somatic synapses contain a few spherical synaptic vesicles and have symmetric membrane densities. Flattened synaptic vesicles were first found on day 10 and increased throughout development. Although a few axo-somatic synapses with asymmetric membrane specializations were found at practically all stages, the symmetric type was always in the majority. An attempt was made to relate these observations with physiological, behavioral and neuroembryological findings from birds and other forms. For example, the fact that axo-dendritic synapses always appear prior to axo-somatic contacts would seem to rule out the role of somatic synapsesin the initial induction of dendritic growth in the spinal cord.     

GABA immunoreactive axons and growth cones in the developing chicken optic nerve and tract.

Immunohistochemical studies of the chicken embryo optic tract using an antibody to gamma-aminobutyric acid (GABA) reveal that the tract is initially free of GABA immunoreactive axons. During the second week of incubation, GABA+ axons appear in the tract, chiasm, and optic nerve. The number of GABA+ axons in the optic nerve increases through E18, although few are recognizable after hatching. Detailed staining of GABA+ growth cones confirmed that virtually all the GABA+ axons in the optic nerve were growing toward the retina. Taken together, the findings suggest that the GABA+ axons in the chiasm and nerve are largely a transient extension of the GABA+ optic tract cells, the tectogeniculate projection, or both.     

The environment of axonal migration in the developing chick retina: a scanning electron microscopic (SEM) study.

We have made a SEM study of the basal intercellular spaces of the retina in chick embryos of different developmental stages. Since this is the environment where optic axons grow, the structural characteristics of this region might play some role in the orientation of axonal migration towards the choroid fissure. The basal region of undifferentiated retinas is formed by the vitreal expansions of neuroepithelial cells. In pre-axonal stages, the intercellular spaces between these expansions do not show any preferential orientation towards the fissure. The growth cones of ganglion cell axons appear in an apicobasal direction and turn towards the fissure immediately beneath the vitreal surface. Fasciculation is an early event during development and, in the more advanced stages, the vitreal expansions from retinal cells are placed in rows following the same orientation as the axon bundles. These observations are discussed in relationship to current hypotheses on axonal migration and orientation.     

A morphological and morphometric analysis of the optic nerve in the hypothyroid rat.

This investigation was designed to morphologically evaluate the effects of hypothyroidism on the development of myelin and axons in the rat optic nerve. Four pups from each group of normal and propylthiouracil-induced hypothyroid rats were sacrificed at 14, 21, 28, and 35 postnatal days. Optic nerves were studied by both light and electron microscopes. The hypothyroid animals had significantly reduced body and brain weights compared to those of their age-matched controls. In the hypothyroid animals, the cross-sectional area of the optic nerve, the fiber density, and fiber occupancy were significantly diminished compared to those of the controls. The mean individual fiber size was unaffected. However, the relationship between the total axonal area to myelin thickness was similar in the control and experimental groups, implying that the feedback mechanism between myelinating cells and axons was not affected by hypothyroidism. Thus, this study indicates that the principal insult of neonatal hypothyroidism results in a delay in myelin acquisition of myelinated fibers, resulting in diminished cross-sectional area of the optic nerve, fiber density, and fiber occupancy.     

Distribution of uncrossed axons along the course of the optic nerve and chiasm of rodents

The distribution of the ipsilaterally projecting population of retinofugal axons has been analyzed following injections of horseradish peroxidase (HRP) into the optic tract of adult hamsters and rats to determine whether the topographical segregation of the cells of origin seen in the retina is maintained by their axons throughout the course of the optic nerve and chiasm. Axons are limited to a roughly appropriate topographic location within the intraorbital course of the nerve but this organization changes at levels progressively closer to the optic chiasm. Immediately rostral to the chiasm labelled profiles are found dispersed across most of the cross-sectional area of the nerve. This dispersal is maintained within the region of the optic chiasm where a complex rearrangement of ipsilaterally projecting axons takes place. The results show that axons are not retinotopically organized along the entire length of the optic nerve. The order of axons changes along the course of the nerve and in the optic chiasm. The change seen within the intracranial course may indicate a chronotopic re-sorting of axons prior to the optic tract where the organization of axons has previously been interpreted as a map of time of axon arrival.     

A quantitative morphological study of interstrain variation in the developing rat optic nerve

Groups of pigmented (Black and White Hooded Lister) and albino (Sprague Dawley) rats were killed at 7, 15, and 25 postnatal days of age. Their optic nerves were embedded in resin suitable for both light and electron microscopy. Quantitative stereological procedures were used to estimate total fibre number and the degree of myelination in the optic nerves at the various ages. At 7 days of age, both albino and pigmented rats had about 220,000 optic nerve fibres. By 15 days, both strains showed a reduction of some 30,000 fibres. This fibre loss continued in both strains after 15 days of age, but more rapidly in the pigmented strain. At 25 days of age, pigmented rats had 72,371 +/- 7,244 fibres, whilst albino rats had 102,681 +/- 4,138 fibres (P less than .01). More than 99.5% of optic nerve axons in both strains were unmyelinated at 7 days of age. By 25 days of age about 90% of all remaining fibres were myelinated in both strains. The mean diameter of the myelinated axons was estimated to be between 0.59 and 0.65 micron in all animals, there being no significant age or strain differences. In contrast, the mean diameter of nonmyelinated axons increased significantly with age. This increase was greater for albino than pigmented rats, such that by 25 days of age the respective values were 0.49 micron and 0.42 micron (P less than .05).     

A freeze-fracture study on the developing satellite cells of spinal ganglia in the chick embryo

A freeze-fracture analysis of the satellite cells of spinal ganglia of the chick embryo was performed in 8 successive stages of development, from the 5th incubation day to hatching. The characteristic laminar disposition of the cells were first observed on the 7th day. Tight junctions were found at the 20th incubation day. Small groups or irregular aggregates of particles, but not gap junctions, were described on the 7th and 8th days. Pinocytotic vesicles were pointed out in the different stages considered.     

Changes in the numbers of retinal ganglion cells and optic nerve axons in the developing albino rabbit

In albino rabbits aged from the 16th postconceptional day (16PCD) to adulthood, the number of axons in the optic nerves were estimated from sample areas totalling 1-12% of the cross-sectional area of the nerve. On the 16PCD there are about 20,000 axons in the optic stalk. The number of axons in the retrobulbar part of the optic nerve reaches a peak value of 766,000 on the 23PCD, and then decreases to about 350,000 by the 32PCD (the day of birth). The number of axons does not change between the 32PCD and 50PCD, but thereafter it slowly decreases, reaching the adult number (294,000) by the 84PCD. A similar trend is apparent in pigmented animals. Thus, on the 25PCD there are 736,000 axons in the retrobulbar part of the optic nerve and the number decreases to 428,000 by the 31PCD. In the adult pigmented rabbit there are 280,000 axons in the optic nerve. In animals younger than the 32PCD, growth cones are present, and the number of axons in the prechiasmal part of the optic nerve was 8-22% lower than in the retrobulbar part of the same nerve. These observations suggest that there is a continued outgrowth of axons from the eye towards the target nuclei. By the 32PCD, the numbers of axons in the retrobulbar and prechiasmal parts of the nerve were very similar, suggesting that by this age all axons had reached the chiasm. The numbers of retinal ganglion cells (RGCs) labelled by massive injections of horseradish peroxidase into the retino-recipient nuclei were estimated in albino rabbits aged from the 24PCD to adulthood. RGCs were counted in evenly spaced sample areas totalling 4-11% of the retinal area. On the 24PCD, the number of labelled RGCs (500,000) was lower than the number of axons in the optic nerve (probably because not all RGC axons had reached their target nuclei by this age). However, by the 27PCD the number of labelled RGCs (550,000) was very similar to the number of prechiasmal axons (568,000). At all ages thereafter, the numbers of both RGCs and axons were very similar, with adult RGC numbers (about 291,000) being reached by the 85PCD. We conclude that axon loss in the rabbit optic nerve after the 27PCD is almost certainly due to the elimination (presumably death) of the parent RGCs, and we suggest that RGC death is also the most likely cause of axon loss prior to the 27PCD.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cell death in the developing chick optic tectum

The degeneration and death of early developing neuroblasts in the optic tectum of chick embryo during normal development have been investigated by electron microscopy.