Influence of a 30-day spaceflight on the structure of motoneurons of the trochlear nerve nucleus in mice.

During spaceflight and immediately after it, adaptive neuroplastic changes occur in the sensorimotor structures of the central nervous system, which are associated with changes of mainly vestibular and visual signals. It is known that the movement of the eyeball in the vertical direction is carried out by muscles that are innervated by the trochlear nerve (CN IV) and the oculomotor nerve (CN III). To elucidate the cellular processes underlying the atypical vertical nystagmus that occurs under microgravity conditions, it seems necessary to study the state of these nuclei in animals in more detail after prolonged space flights. We carried out a qualitative and quantitative light-optical and ultrastructural analysis of the nuclei of the trochlear nerve in mice after a 30-day flight on the Bion-M1 biosatellite. As a result, it was shown that the dendrites of motoneurons in the nucleus of the trochlear nerve significantly reorganized their geometry and orientation under microgravity conditions. The number of dendritic branches was increased, possibly in order to amplify the reduced signal flow. To ensure such plastic changes, the number and size of mitochondria in the soma of motoneurons and in axons coming from the vestibular structures increased. Thus, the main role in the adaptation of the trochlear nucleus to microgravity conditions, apparently, belongs to the dendrites of motoneurons, which rearrange their structure and function to enhance the flow of sensory information. These results complement our knowledge of the causes of atypical nystagmus in microgravity.

In Vivo and Ex Vivo Comprehensive Evaluation of Corneal Reinnervation in Eyes Neurotized With Contralateral Supratrochlear and Supraorbital Nerves.

PURPOSE: To use an automated morphometric analysis system of in vivo confocal microscopy (IVCM) images for evaluating reinnervation occurring at the subbasal nerve plexus (SNP) after direct corneal neurotization (DCN) and to further report neurophysiological and histopathological findings. METHODS: Prospective interventional case series including 3 eyes with neurotrophic keratitis that underwent DCN. Deep anterior lamellar keratoplasty was performed 18 months after DCN in patient 1. The following evaluations were performed before and at 3, 6, and 12 months after DCN: clinical evolution of keratitis; corneal sensitivity; IVCM images of the SNP analyzed with "ACCMetrics;" neurophysiological study of corneal reflex. Protein gene product 9.5 immunofluorescence staining assay and transmission electron microscopy were conducted on the neurotized button excised during deep anterior lamellar keratoplasty. RESULTS: Complete healing was obtained in all patients by 3 months postoperatively. Corneal sensitivity was absent preoperatively in all eyes and improved after surgery, reaching an average value of 30 mm 1 year postoperatively. The corneal SNP was not visible at IVCM in any of the cases preoperatively and became visible by 3 months postoperatively, showing IVCM metrics comparable to normal contralateral eyes at 1 year. In all cases, neurophysiological evaluation showed a partial recovery of the electrical activity of the cornea. In patient 1, protein gene product (PGP) 9.5 staining of neurotized cornea showed nerve fascicles at the SNP, whereas transmission electron microscopy showed amyelinic nerve axons and nerve endings. CONCLUSIONS: The corneal SNP exhibited IVCM metrics comparable to the normal contralateral eye 1 year after DCN. Ex vivo histopathological assessment of neurotized corneas confirmed the presence of nerves with normal ultrastructure.

The trochlear nerve: microanatomic and endoscopic study.

The purpose of the present study was to analyze the relationships of the trochlear nerve with the surrounding structures through both endoscopic and microscopic perspectives. The aim was to assess the anatomy of the nerve and to carry out a thorough description of its entire course. A comprehensive anatomically and clinically oriented classification of its different segments is proposed. Forty human cadaveric fixed heads (20 specimens) were used for the dissection. The arterial and venous systems were injected with red and blue colored latex, respectively, in the transcranial dissection. For illustrative purposes, the arterial vessels were injected alone in endoscopic endonasal procedures. A CT scan was carried out on every head. Median supracerebellar infratentorial, subtemporal, fronto-temporo-orbito-zygomatic, and endoscopic endonasal transsphenoidal approaches were performed to expose the entire pathway of the nerve. A navigation system was used during the dissection process to perform the measurements and postoperatively to reconstruct, using dedicated software, a three-dimensional model of the different segments of the nerve. The trochlear nerve was divided into five segments: cisternal, tentorial, cavernous, fissural, and orbital. Detailed and comprehensive examination of the basic anatomical relationships through the view of transcranial, endoscope-assisted, and pure endoscopic endonasal approaches was achieved. As a result of a thorough study of its intra- and extradural pathways, an anatomic-, surgically, and clinically based classification of the trochlear nerve is proposed. Precise knowledge of the involved surgical anatomy is essential to safely access the supracerebellar region, middle fossa, parasellar area, and orbit.

Structural and ultrastructural study of the intracranial portion of the oculomotor, trochlear and abducent nerves in dog.

The right intracranial portion of the oculomotor, trochlear and abducent nerves were removed from six adult German shepherd dogs and analysed by light and electron microscopy. In all cases the nerve sectional area was calculated. Unmyelinated and myelinated fibres were analysed and number, diameter and cross-sectional area were calculated. In myelinated fibres, also calculated were the corresponding axon area and diameter, and myelin sheath thickness. The mean number of myelinated fibres was 8543.50 +/- 1231.85 being the unmyelinated 1402 +/- 241.58 in the oculomotor nerve; 1509 +/- 223.17 and 287.67 +/- 72.28 in the trochlear nerve and 2473.00 +/- 211.41 and 231.25 +/- 92.67 respectively in the abducent. The mean diameter was 10.23 +/- 0.68 microm in myelinated and 0.43 +/- 0.21 for unmyelinated in oculomotor nerve, 10.53 +/- 0.55 microm and 0.33 +/- 0.04 for the trochlear, and 10.45 +/- 1.27 microm and 0.47 +/- 0.09 in the abducent nerve respectively. This study reveals that oculomotor, trochlear and abducent nerves of the dog show structural and ultra-structural features similar to the same nerves in other species.

Motor axon guidance of the mammalian trochlear and phrenic nerves: dependence on the netrin receptor Unc5c and modifier loci.

Netrin signaling is important to guide migrating neurons and axons in many systems. Experiments with vertebrate CNS explants suggested netrin is bifunctional, attracting some axons and repelling others. Netrin1-expressing cells attracted spinal commissural axons and repelled trochlear cranial nerve axons in these experiments. Subsequent genetic studies demonstrated that multiple axon types, including those of the spinal commissural neurons, are attracted to netrin in vivo; however, an in vivo role for netrin signaling in trochlear nerve repulsion has not been observed. Here, we demonstrate that mice with a null mutation in the netrin receptor Unc5c on the inbred C57BL/6J (B6) genetic background have ventral/ipsilateral trochlear nerve misprojections. These misprojections are attenuated on a hybrid B6 x SJL background. In addition, B6.Unc5c(-/-) mice die as neonates of apparent respiratory distress and have incomplete phrenic nerve innervation of the diaphragm muscle. Neither the trochlear nerve misprojections nor the phrenic nerve phenotype was observed in B6 embryos lacking the netrin receptors DCC or Neogenin1, or the ligand netrin1, indicating these signaling molecules are dispensable for guidance of these axons. Like the trochlear nerve, the phrenic nerve phenotype is modified in a B6 x SJL hybrid background. To identify these modifier loci, we performed genome scans of the hybrid Unc5c(-/-) mice and found a major SJL-derived suppressor locus on Chromosome 17. Our results provide the first evidence that genes involved in netrin signaling are necessary for proper mammalian spinal motor axon development and trochlear axon guidance. In addition, they demonstrate the importance of modifier genes in vertebrate axonal guidance.

Estudio estructural y ultraestructural de la porción intracraneal del nervio troclear del perro / Structural and ultrastructural study the intracranial portion of the trochlear nerve in the dog

En el presente trabajo se han extraído, de seis perros adultos, de 40-50 kilogramos de peso, los nervios trocleares derechos en su porción intracraneal. Los nervios fueron analizados con microscopia óptica y microscopia electrónica. Hay que destacar la presencia de fibras no mielinizadas en todos los nervios analizados. De las fibras mielinizadas hemos determinado su número, diámetro, área y densidad , así como el área y el diámetro del axón correspondiente y el grosor de la vaina de mielina. El número medio de fibras mielínicas contabilizadas fue de 1482,25. El histograma de frecuencias relativo al grosor de la vaina de mielina y al tamaño fibrilar muestran una distribución bimodal, siendo 10,8 μm el valor medio de discriminación de las dos poblaciones fibrilares. Se aprecia una proporción similar de fibras grandes (47,45%) y pequeñas (52,55%). La población de fibras pequeñas presentan un tamaño medio de 6,44 μm y ocupan un 12,10% de la superficie total del nervio, por el contrario las fibras grandes, con un tamaño medio de 15,34 μm ocupan el 60,20% del nervio (AU)

In the present investigation, the right intracranial portion of the trochlear nerves was removed from six adult dogs weighing 40-50 kg and analyzed by light and electron microscopy. A significant number of unmyelinated fibers were observed in the analyzed nerves. Number, diameter, area and density of myelinated fibers were determined as were corresponding axon area and diameter and myelin sheath thickness. Myelinated fibers count produced a mean of 1482,25. Frequency histograms of myelin sheath thickness and fiber size show a bimodal distribution being 10,8 μm the mean value of discrimination of the two populations. A similar proportion of large (47,45%) and small fibers (52,55%) is appraised. The small fibers population was present in a size mean of 6,44 μm and occupies a 12,10% of the total surface of the nerve, on the contrary the large fibers, with size mean of 15,34 μm ocupy a 60,20% of the nerve (AU)

Intracranial portion of the trochlear nerve and dorsal oblique muscle composition in dog: a structural and ultrastructural study.

In the present investigation the right intracranial portion of the trochlear nerves and dorsal oblique muscle of the right ocular globe were removed from six adult dogs and analyzed by light and electron microscopy. Unmyelinated fibers were observed in the analyzed nerves. The number, diameter, area, and density of myelinated fibers were determined, as were corresponding axon area and diameter and myelin sheath thickness. Frequency histograms of myelin sheath thickness and fiber size show a bimodal distribution with a similar proportion of large and small fibers. Muscle samples were taken from the central portion of the muscle belly, subsequently frozen, cut, and stained with m-ATPase at pH 4.6. Fibers were classified as Type 1 or Type 2 according to their reaction to the m-ATPase and detailed morphologic and morphometric studies were made. The muscles showed two clearly distinct layers, a central layer and a peripheral layer, chiefly composed of Type 2 fibers. The fibers in the central layer were larger in size than those in the peripheral layer.

Axon-glial relations during regeneration of axons in the adult rat anterior medullary velum.

The anterior medullary velum (AMV) of adult Wistar rats was lesioned in the midsagittal plane, transecting all decussating axons including those of the central projection of the IVth nerve. At selected times up to 200 days after transection, the degenerative and regenerative responses of axons and glia were analyzed using transmission and scanning electron microscopy and immunohistochemistry. In particular, both the capacity of oligodendrocytes to remyelinate regenerated fibers and the stability of the CNS/PNS junctional zone of the IVth nerve rootlet were documented. Transected central AMV axons exhibited four patterns of fiber regeneration in which fibers grew: rostrocaudally in the reactive paralesion neuropil (Group 1); randomly within the AMV (Group 2); into the ipsilateral IVth nerve rootlet, after turning at the lesion edge and growing recurrently through the old degenerated contralateral central trochlear nerve trajectory (Group 3); and ectopically through paralesion tears in the ependyma onto the surface of the IVth ventricle (Group 4). Group 1-3 axons regenerated unperturbed through degenerating central myelin, reactive astrocytes, oligodendrocytes, microglia, and large accumulations of hematogenous macrophages. Only Group 3 axons survived long term in significant numbers, and all became myelinated by oligodendrocytes, ultimately establishing thin sheaths with relatively normal nodal gaps and intersegmental myelin sheath lengths. Schwann cells at the CNS/PNS junction of the IVth nerve rootlet did not invade the CNS, but astrocyte processes grew across the junction into the PNS portion of the IVth nerve. The basal lamina of the junctional glia limitans remained stable throughout the experimental period.

Utricular input to cat extraocular motoneurons.

Intracellular recordings from 200 identified extraocular motoneurons in the bilateral III, IV and VI cranial nuclei were studied to determine the connectivities between the utricular nerve and the extraocular motoneurons in cats. Stimulating electrodes were placed within the left utricular nerve, while other branches of the vestibular nerve were removed. Monosynaptic and disynaptic connections between the utricular nerve and the ipsilateral abducens motoneurons and interneurons were recorded as described previously. Stimulation of the utricular nerve evoked longer latency depolarizing and hyperpolarizing potentials in contra- and ipsilateral medial rectus motoneurons, respectively. Depolarizing and hyperpolarizing potentials with longer latencies were also recorded in the ipsilateral inferior oblique and contralateral trochlear motoneurons. The short and longer latency circuits between the utricular nerve and extraocular motoneurons may play a role in stabilizing the retinal image during head tilt and horizontal linear acceleration.

Regenerative axonal sprouting in the cat trochlear nerve.

Following peripheral trochlear nerve axotomy in the cat, the normal number of myelinated axons is restored despite significant motor neuron death, suggesting regulation of the number of myelinated axons in the regenerated nerve. In this study we used light and electron microscopy to examine the production and maintenance of axonal sprouts at different locations in the nerve and at different postoperative intervals. Despite proliferative sprouting and an overproduction of nonmyelinated axons in the regenerating trochlear nerve, the number of myelinated axons was strictly regulated. Only approximately 1,000 regenerated axons were eventually remyelinated, but many nonmyelinated axons were still present 6-8 months postaxotomy. Regenerated axons were remyelinated in a proximal-to-distal direction between 3 and 4 weeks postaxotomy. We also examined the maturation of regenerated myelinated axons by measuring axon diameter and myelin index (an expression of myelin thickness). Mean myelinated axon diameter remained significantly below normal in long-term regenerated nerves. Mean myelin index was not different from normal at 4 weeks postaxotomy but was significantly decreased at long postoperative intervals, reflecting a slightly thicker myelin sheath relative to the axon diameter. This relative increase in mean myelin thickness could serve to restore normal conduction velocity despite the decrease in mean axon diameter. We suggest that the regulation of the number of myelinated axons at the normal number despite cell death and the increase in mean myelin thickness may both be compensatory mechanisms that function to restore preoperative conditions and maximize functional recovery.

Regeneration of axons into the trochlear rootlet after anterior medullary lesions in the rat is specific for ipsilateral IVth nerve motoneurones.

The fibre projection from the IVth nerve nucleus to the superior oblique muscle was determined quantitatively in the normal rat by defining fibre numbers in transverse sections of the IVth nerve, and neurone numbers after retrograde labelling by horseradish peroxidase (HRP) injection into the muscle. There were 183 +/- 27 (S.E.) labelled neurones in the nucleus contralateral to the injected muscle and only 2 +/- 1 ipsilateral. The ipsilateral fibre number was 234 +/- 7 and the cell/axon ratio 0.8 +/- 0.1. Extensive analysis of all HRP retrogradely labelled material revealed no central fibre contribution to the IVth nerve other than from neurones resident in the trochlear nucleus. The central portion of the trochlear nerve tract was severed at its point of decussation in the anterior medullary velum. Ninety days after lesion, 10 +/- 4 (6% of control) neurones were labelled in the ipsilateral trochlear nucleus; none were labelled in the contralateral nucleus or in any other part of the midbrain, pons, medulla, or cerebellum. The number of myelinated fibres in the IVth nerve had decreased to 21 +/- 5 (9% of control) so that the cell/axon ratio was 0.4 +/- 0.2, thus suggesting that a single motoneurone has more fibres after lesion. In electron micrographs of the IVth nerve, larger than normal numbers of unmyelinated fibres were seen. Many myelinated fibres displayed signs of abnormal myelination. After regeneration, the projection was exclusively ipsilateral and not crossed as in the normal. These findings establish that there is a high degree of specificity after regeneration since no myelinated central nervous system axons other than trochlear fibres select the IVth nerve root as a trajectory over which to regenerate.

Sequential double labelling with different fluorescent dyes coupled to dextran amines as a tool to estimate the accuracy of tracer application and of regeneration.

We present a technique to estimate the accuracy of a given application procedure for neuronal tracers. In a second series of animals we used this technique for the estimation of successful regeneration of peripheral nerves. Dextran amine coupled to rhodamine was applied to the cut trochlar nerve in Xenopus tadpoles. To assess the accuracy of tracer application, experiments were done in which a second dye, dextran amine coupled to fluorescein, was applied after 1 day proximal to the first dye. More then 90% of all trochlear motoneurons were doubly labelled after this procedure. Their total numbers were not significantly different from numbers obtained after single labelling with HRP in a comparable age group. To assess success of regeneration after 5 and 8 days, the second application of fluorescein dextran amine was distal to the first application side. Statistically significant differences suggest incomplete regeneration of many neurons. After 42 days the numbers of singly and doubly labelled motoneurons was in the same proportion as before regeneration. This suggests that about 90% of the surviving motoneurons had successfully regenerated back to the periphery.

Synapse formation on trochlear motor neurons in relation to naturally occurring cell death during development.

About half of the trochlear motor neurons die during the course of normal development. The present study was undertaken to determine whether the afferent synapses form before the onset of motor neuron death and also to determine whether the number of synapses differs between the healthy and degenerating trochlear motor neurons. Brains of duck embryos from days 10 to 20 were prepared for quantitative electron microscopical observations on synaptogenesis. Results indicate that synapses form on the trochlear motor neuron soma before cell death begins suggesting that afferent input is in a position to exert an influence on survival or death of motor neurons. There were no significant differences in the number of synapses between the healthy and dying neurons during the period of cell death. This observation suggests that the mechanism by which afferent synapses could be involved in neuron survival or death is not related to the number of synapses on the cell soma. The number of synapses on the cell process, synaptic transmission and/or molecules released at the synapses are likely candidates for the mechanism of action of afferent input.

Axon-myelin relationships in rat cranial nerves III, IV, and VI: a morphometric study of large- and small-fibre classes.

The primary objectives of this study were to determine (1) if quantitative axon-myelin relationships are similar for large- and for small-fibre classes within individual nerves and (2) if the same axon-myelin relationships hold for equivalent fibre classes in closely similar nerves. The oculomotor, trochlear, and abducent nerves of the rat were examined since they each contain distinct large- and small-fibre classes and are similar in a wide range of anatomical and developmental respects. Accordingly, morphometric analyses of axon-myelin relationships were performed separately on large and small fibres of each of the three nerves. Within each nerve, the setting of the relationship between the two parameters was found to be different for the two fibre classes: Scatterplots relating sheath thickness to axon perimeter for large fibres were shifted upwards relative to those for small fibres. These differences were also reflected in the positions of the regression lines fitted to the plots and in the g-ratios. Significant differences were found between nerves in relation to their large fibres: Those of the abducent nerve had significantly thicker sheaths, those of the oculomotor nerve had significantly smaller axon perimeters, and the myelin sheath-axon perimeter relationship of the abducent nerve differed significantly from that of the other two. This study therefore shows that morphometric axon-myelin relationships may differ significantly between equivalent fibre classes of nerves that are closely similar in respect of morphological class, central origin, peripheral distribution, developmental environment, and function.

Fibre composition of the feline trochlear and abducens nerves.

Cat trochlear and abducens nerves were studied by electron microscopy at two different levels. Five mm peripheral to the exit from the brainstem, the average number of myelinated axons is 965 in the trochlear nerve and 1901 in the abducens nerve. The size spectrum is unimodal and small myelinated axons predominate. Both nerves contain 16% unmyelinated axons at this level. At the PNS/CNS transition, the nerve fascicles contain few unmyelinated axons, but bundles of such axons are present in the adjacent pia mater. We suggest that the trochlear and abducens nerves may channel unmyelinated sensory and/or autonomic axons to the leptomeningeal blood vessels and the pia mater of the brainstem.

The trochlear motoneurons of lampreys (Lampetra fluviatilis): location, morphology and numbers as revealed with horseradish peroxidase.

The cells of origin of the trochlear nerve of Lampetra fluviatilis have been labelled with horseradish peroxidase (HRP) in order to compare the location and morphology of trochlear motoneurons with those of other vertebrates and to gain insight into the phylogenetic changes of the trochlear system. About 126 bipolar and tripolar trochlear motoneuron perikarya are found in a dorsal tegmental position close to the trochlear root. Only approximately 16% of the labelled cells are on the ipsilateral side of the brain, i.e. they lie predominantly contralateral as in gnathostome vertebrates. Dorsally directed dendrites reach the area of lateral-line and retinofugal fibres, and may establish functional contacts. In addition, each motoneuron has a ventral dendrite that extends towards the fasciculus longitudinalis medialis and to the ventral tegmentum. The dendrites branch close to the oculomotor root. Lampreys show a low muscle fibre to motoneuron ratio (4.5:1), i.e., they resemble amniotic vertebrates more than other anamniotic vertebrates. These data demonstrate both closer resemblance and larger differences of cyclostome and gnathostome trochlear motoneurons than previously suggested.

Degenerative and regenerative changes in the trochlear nerve of goldfish.

The features of unlesioned and lesioned trochlear nerves of goldfish have been examined electron microscopically. Lesioned nerves were studied between 1 and 107 days after cutting or crushing the nerve. Unlesioned nerves contained, on average, 77 myelinated axons and 19 unmyelinated axons. The latter were found in 1-2 fascicles per nerve. A basal lamina surrounded each myelinated axon and fascicle of unmyelinated axons. The numbers of myelinated axons, fascicles of unmyelinated axons and basal laminae varied by less than 5% over the intraorbital extramuscular segment of the nerve. Following interruption of the nerve, by either cutting or crushing, all of the axons and their myelin sheaths began to degenerate by 4 days in the distal nerve-stump. Both abnormally electron-dense and electron-lucent axons were observed. Both Schwann cells and macrophages appeared to phagocytose the myelin sheaths. Following a lesion, the Schwann cells and their basal laminae persisted in the distal nerve-stump. In crushed nerves, the basal laminae surrounding myelinated axons formed 97%, on average, of the Schwann tubes in the distal stump. The perimeters of the basal laminae were of similar size to those in the proximal stump, at least for the first 8 days after crush. In crushed nerves, single myelinated axons in the proximal nerve-stump gave rise to multiple sprouts, some of which reached the site of crush by 2 days, the distal stump by 4 days and the superior oblique muscle by 8 days. The regeneration of the unmyelinated axons was not examined. In both crushed and transected nerves, nearly all of the sprouts in the proximal and distal stumps were found within the basal laminae of Schwann cells, even though the spouts were disorganized in the transected region where there were no basal laminae. The growth cones of the regenerating axons were always found apposed to the inner surface of the basal laminae, which may have provided an adhesive substrate that directed their growth. Terminal sprouts from the ends of myelinated axons in the proximal stump accounted for the majority of the regenerating axons in the distal stump, as only a few collateral sprouts were found in the proximal stump, and only a small amount of axonal branching was found within the distal stump itself. The largest axons in the distal stump were remyelinated first, and the number of remyelinated axons increased progressively between 8 and 31 days after crush, at which time there were about twice as many as in unlesioned nerves.(ABSTRACT TRUNCATED AT 400 WORDS)

Decrease in acetylcholine receptor number correlated with increased naturally occurring trochlear motor neuron death during development.

It has been observed that daily application of neostigmine onto the chorioallantoic membrane drastically reduced the total number of acetylcholine receptors in the superior oblique muscle of duck embryos. Here the effects of neostigmine on the magnitude of naturally occurring death of trochlear motor neurons during embryonic development were investigated. There was an enhanced loss of neurons in the neostigmine-treated embryos. Neostigmine neither affected the initial production of normal numbers of motor neurons nor had any direct toxic effect on their ultrastructure. The decrease in muscle activity did not always correlate with increased motor neuron survival. There may be a relationship between acetylcholine receptor distribution and naturally occurring neuronal death.