Orthograde Axonal Transport Research Articles

Clinically Observable Ocular Manifestations of Axonal Transport

This report illustrates and provides a novel explanation for post-trabeculectomy improvements in the visual field, cup disc contours, and apparent deepening of an arcuate nerve fiber layer (NFL) defect after trabeculectomy for open-angle glaucoma. These changes are all plausible manifestations of recovered axonal transport and thickening retina, previously thinned by elevated intraocular pressure (IOP). Serial pre-and post-operative clinical fundus photos in case 1(A,B) demonstrate increased prominence of an inferior temporal arcuate nerve fiber layer defect, improved cup disc ratio, and visual field following eye pressure lowering by + 50% after trabeculectomy. Case 2 (C,D) also demonstrates obvious cup disc improvement in post-operative photos with associated improvement in visual field after trabeculectomy and lowering IOP by + 30%. We suggest that elevated IOP suppresses primarily orthograde axonal transport resulting in nerve fiber layer (NFL) thinning that can recover back to normal thickness when IOP is surgically lowered by the magnitude achieved in these two examples.

Axonal Transport in Coats’ Disease and Congenital Oculodentodigital Syndrome

Introduction: Axoplasmic transport blockade has been demonstrated in acute experimental glaucoma in animals but has not been documented in childhood glaucoma. The purpose of this study was to detail findings and axoplasmic transport status in one eye of a two-year old male with Coat’s disease using the two eyes from a 10-month-old female with Oculodentodigital Syndrome (ODDS) as controls for background axoplasmic transport. We document for the first-time axonal transport block in the lamina cribrosa (LC) in a blind and painful right eye associated with Coats’ disease, (Case 1). Enucleation was done for suspected retinoblastoma without imaging. Histopathology demonstrated myriad macrophages migrated from the vitreous infiltrate into the anterior chamber and meshwork explaining the glaucoma. Two eyes from Case 2, diagnosed at autopsy as having congenital Oculodentodigital Syndrome (ODDS), with a history of surgically normalized intraocular pressures were included as controls for the Coats’ case immunohistochemistry (IHC) and to report additional findings in this rare disorder. Methods: We reviewed available history and performed histopathology on one enucleated Coats’ globe and two additional autopsy eyes from an ODDS case with routine staining of paraffin-processed tissues and amyloid precursor protein (APP-A4) IHC, the only available IHC marker for orthograde axonal transport in human surgical or autopsy tissues. The two eyes from the 10-month-old infant with ODDS had normal IOPs following trabeculotomies at five months of age. Results: In addition to typical pathology for Coats’ the lamina cribrosa portion of the optic nerve from Case 1 demonstrated marked orthograde axonal transport block. Both eyes from Case 2 with ODDS had a history of congenital glaucoma treated medically before bilateral trabeculotomies at five months-age with normalization of IOPs. Descemet scrolls found OD were judged residual to goniotomy. Both globes from Case 2 demonstrated multiple anomalies previously reported in ODDS including microphthalmos, optic disc dysplasia OD, and features of persistent hyperplastic primary vitreous (PHPV) OU. The right eye also demonstrated spontaneous retinal detachment prompting an exam under general anaesthesia at 10-months age when, during induction, she expired with malignant tachycardia. Retinal ganglion cells (RCGs) labeled with APP-A4 OU, but optic nerve LC axons demonstrated only background accumulations of the IHC marker. Conclusions: Orthograde axonal transport was interrupted in the LC of the Coats’ nerve due to secondary inflammatory glaucoma. The angle remained open histologically in-spite-of-early neovascularization and pupillary margin ectropion. Block of orthograde axonal transport in the LC has been well established as the site of initial optic nerve damage in glaucoma in experimental studies and this report reinforces its importance to the pathophysiology of glaucoma optic nerve damage. The primary importance of the two ODDS globes for our report was as comparison controls for background APP-A4 staining in the Coats’ case. Our additional findings in the ODDS eyes suggest delayed spontaneous retinal detachment, and lens epithelial abnormalities are additional anomalies to add to the list of known malformations in this rare disorder. Case 2 also serves as a caution in ODDS regarding vulnerability to combinations of medications when general anaesthesia is contemplated.

Amyloid Precursor Protein in Abusive Head Trauma Suspects

To study orthograde axonal transport with amyloid precursor protein (APP-A4) immunohistochemistry (IHC) in the retina and lamina cribrosa (LC) portion of the optic nerve in abusive head trauma (AHT) suspects. Retrospective, case-control study. Seventy-two eyes from suspected AHT victims referred by the Los Angeles Coroner and control eyes from nontraumatized infants were included. IHC was conducted using University of California, Irvine (UCI), Lab Medicine Department's standard protocol and results analyzed by light microcopy after paraffin processing. Quantitation of LC APP-A4 block was estimated in 21 cases with known survival using MetaMorph, a proprietary biomicroscopy imaging software. The presence or absence of APP-A4 label accumulations in retinal ganglion cells, nerve fiber layer at the disc margin, and in LC axonal bundles were compared to matching tissues from nontraumatized control eyes with only background staining. Among the globes from AHT suspects with nerve heads available for study, 94% were positive for LC accumulation of marker. Among suspect AHT cases with known survival after injury of 1 to 1588 days, most demonstrated LC APP-A4 accumulations. Our findings reinforce a recent publication based on APP-A4 IHC that demonstrated similar orthograde axonal transport block in the LC in children with AHT and recommend that intraocular pressures be recorded and addressed in these patients.

Selective Early Glial Reactivity in the Visual Pathway Precedes Axonal Loss, Following Short-Term Cerebrospinal Fluid Pressure Reduction.

To examine the early glial reactivity and neuron damage in response to short-term cerebrospinal fluid pressure (CSFp) reduction, as compared with intraocular pressure (IOP) elevation. The experiment included 54 male Sprague-Dawley rats with elevated translaminar cribrosa pressure difference (TLPD), defined as IOP minus CSFp. These rats underwent either continuous CSF drainage for 6 hours (n = 18), or unilateral IOP elevation to 40 mm Hg for 6 hours (n = 18). For control, 18 normal rats were anesthetized for 6 hours. Orthograde axonal transport was examined by intravitreal injection of 3% rhodamine-β-isothiocyanate. We also used transmission electron microscopy to display the ultrastructural features of retinal ganglion cell axons in the optic nerve head. Early glial reactivity in the retina, lateral geniculate nucleus (LGN), and superior colliculus (SC) was detected by immunostaining and Western blot for the glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS). We also observed the glial reactivity in the inferior colliculus and hippocampus to rule out possible influences of CSF dynamics and composition. Anterograde staining with 3% rhodamine-β-isothiocyanate revealed decreased fluorescence intensity of the SC and LGN projected from both lower CSFp and higher IOP eyes. Transmission electron microscopy showed loss of axons from the optic nerve head in the high-IOP group, but not in the low-CSFp group. Compared with the anesthesia control group, GFAP expression was significantly increased in the retina, LGN, and SC, whereas GS expression was only increased in the retina following CSFp reduction. However, their expressions were not significantly elevated in the inferior colliculus and hippocampus. In the high-IOP group, expressions of GFAP and GS were significantly increased in the retina, LGN, and SC. Visual system neurons may be much more sensitive than other nervous tissues. Following short-term CSFp reduction, early glial reactivity may precede axonal loss. Changes of translaminar cribrosa pressure difference in both experimental low-CSFp and high-IOP groups induce selective early glial reactivity. The neuron damage from abnormally low CSFp may be pathogenetically similar to high IOP.

Axonal Transport in the Rat Optic Nerve Following Short-Term Reduction in Cerebrospinal Fluid Pressure or Elevation in Intraocular Pressure.

To examine the influence of short-term reduction in cerebrospinal fluid pressure (CSFP) as compared with short-term elevation in IOP on axonal transport. The study included 111 adult Sprague-Dawley rats. For 6 hours, IOP was unilaterally elevated to 40 mm Hg (IOP40-group; n = 27), IOP was unilaterally increased to a value of 25 mm Hg below the mean blood pressure (PP25-group; n = 27), or CSFP was reduced by continuous aspiration of cerebrospinal fluid (Low-CSFP-group; n = 27). A sham control group (with a trocar in cisterna magna without cerebrospinal fluid release) included 24 rats. The left eyes of the IOP40 study group and PP25 study group served as additional contralateral control group. Orthograde axonal transport was examined by intravitreally injected rhodamine-β-isothiocyanate; retrograde axoplasmic flow was assessed by fluorogold injected into the superior colliculi. At 24 hours after baseline, rhodamine-β-isothiocyanate (RITC) staining intensity of the optic nerve was lower (P < 0.05) in the IOP40-group, PP25-group, and Low-CSFP-group than in the control groups. At 6 hours after the fluorogold injection, fluorogold fluorescence was significantly lower in the IOP40-group, the PP25-group, and the Low-CSFP-group than in the control groups. At 5 days after baseline, the fluorogold fluorescence no longer differed significantly between the IOP40-group or the Low-CSFP-group and the control groups. At 1 week after baseline, retinal ganglion cell density was markedly reduced only in the PP25-group. Both short-term lowering of CSFP and short-term rise in IOP were associated with a disturbance of both the orthograde and retrograde axonal transport. The findings support the notion of an association between abnormally low CSFP and optic nerve damage.

Retinal projections in two crocodilian species, Caiman crocodilus and Crocodylus niloticus.

The retinal projections of Caiman crocodilus and Crocodylus niloticus were investigated by means of the orthograde axonal transport of either rhodamine beta-isothiocyanate or tritiated proline. In these two species, each tracer revealed contralateral retinal projections to three hypothalamic regions (subventricular gray matter, nucleus suprachiasmaticus, and area optica hypothalami lateralis), five thalamic regions (nuclei ovalis, dorsolateralis anterior, ventrolateralis and ventrobasalis, and lateral geniculate complex, of which six subcomponents can be distinguished), six pretectal regions (nuclei posterodorsalis, lentiformis mesencephali, griseus tectalis, geniculatus pretectalis, area optica commissurae posterior and area optica pretectalis lateroventralis), six outermost layers of the optic tectum, and the nucleus opticus tegmenti. Weak ipsilateral retinal projections have been observed in two hypothalamic nuclei and in the nucleus opticus tegmenti. Comparative analysis with other data show that the contralateral retinal projections of crocodiles are considerably more reptilian than avian. Moreover, crocodiles share with birds an extremely poor contingent of ipsilateral retinal projections.

Orthograde axonal transport studies of projections from the zona incerta and pretectum to the basilar pontine nuclei in the rat.

This study employed orthogradely transported axonal tracers to demonstrate, in the rat, projections that reach the basilar pontine nuclei from the zona incerta or pretectal nuclei. Except for the most rostral levels, all subdivisions of the zona incerta give rise to substantive basilar pontine projections. Although some topographic differences exist among the temination patterns of various subdivisions, no clear somatotopically organized scheme is apparent. Most incertopontine axons descend to the basilar pons in association with fibers of the medial lemniscus or crus cerebri and reach ipsilateral ventral and medial pontine gray regions. A sparse number of terminals are evident in the contralateral medial pontine gray. The anterior pretectal axons also descend with the medial lemniscus and crus cerebri to enter exclusively the ipsilateral basilar pons where they terminate most densely in ventral and medial regions. Dual orthograde labeling experiments indicate that some pretectal terminal fields in the pontine gray are shared with incertopontine projections and with afferents from the dorsal column nuclei. This potential convergence of basilar pontine afferent projections is significant in light of 1) the known somatosensory input to the zona incerta and pretectum and 2), the fractured somatotopy of peripheral cutaneous inputs that arrive in the cerebellar cortex via mossy fibers. The present studies also employed electron microscopy to identify synaptic boutons formed by incerto- and pretectopontine axons, and they proved to be remarkably similar. Each is a medium to small-sized bouton that contains spheroidal synaptic vesicles and forms asymmetric membrane specializations. Most incerto- and pretectopontine boutons participate in glomerular synaptic complexes that include a single, centrally located bouton contacted on its perimeter by several types of dendritic profiles including shafts and spine-like appendages. A relatively small number of labeled boutons of either type contacts single, isolated dendritic elements in the neuropil. Taken together, these findings suggest that some basilar pontine neurons might receive convergent inputs from the zona incerta and pretectum as well as other somatosensory-related systems such as the dorsal column nuclei and sensorimotor cortex.

Mechanical sensitivity of regenerating myelinated skin and muscle afferents in the cat.

These experiments describe the responses of myelinated skin and muscle afferent nerve fibres at a neuroma to stretch, local pressure and vibration in the anaesthetised cat. The sural nerve and the nerve supplying the medial gastrocnemius were studied. Neuroma formation was encouraged by placing the cut end of the nerve in a cuff made of synthetic material (Gore-tex). By 6 days after nerve section, the two nerves contained mechanically sensitive afferents. No motor fibres appeared to be mechanically sensitive. Mechanically sensitive sural afferents responded to ramp stretch of the nerve, applied at the cuff, with a single impulse or brief burst of impulses. The majority of gastrocnemius afferents responded to stretch with slowly adapting trains of impulses. Many muscle group II afferents exhibited a steady resting discharge, while group I afferents had an intermittent or bursting resting discharge or were silent. Those group I axons which showed resting activity had a low stretch threshold and were probably Ia fibres. Many of the silent units were also stretch sensitive. It is proposed that the spontaneously active units and silent units with low stretch thresholds were Ia fibres, while silent units with high stretch thresholds were Ib fibres. Both sural and gastrocnemius afferents responded to locally applied vibration. The mean peak response frequency for sural units was 170 Hz (+/- 70 Hz SD). For gastrocnemius units it was 325 Hz (+/- 86 Hz SD). Group I muscle afferents responded to higher frequencies of vibration than group II afferents. In four experiments the nerve was treated at a site a few millimetres proximal to the point of section with the axonal transport blocker colchicine. Twenty-five millimolar colchicine blocked impulse conduction at its point of application. Nevertheless, mechanically sensitive areas developed in the nerve just proximal to the treated region. Ten millimolar colchicine did not block impulse conduction, but led to dispersion of mechanosensitive areas to more proximal regions of the mechanosensitive areas to more proximal regions of the nerve. This result suggests that the disruption of orthograde axonal transport by colchicine leads to development of mechanically sensitive areas in axons further back from their cut ends. Local application of the drugs succinyl choline, tetra-ethyl ammonium and gadolinium had no effect on levels of resting activity or on mechanical sensitivity of afferents in the cuff. The potassium channel blocker 4-aminopyridine, on the other hand, produced an increase in the levels of resting activity and in the stretch responses of afferents.(ABSTRACT TRUNCATED AT 400 WORDS)

Cellular localization of nerve growth factor-like immunoreactivity in adult rat brain: Quantitative and immunohistochemical study

To elucidate the role and the mechanism of action of nerve growth factor in the adult central nervous system, we investigated the localization of nerve growth factor-like immunoreactivity in adult rat brain, both quantitatively and immunohistochemically, using polyclonal anti-nerve growth factor immunoglobulin G. We raised rabbit polyclonal anti-mouse nerve growth factor antibody with an extremely high titer as 10 −9 determined by an enzyme immunoassay. The affinity-purified anti-nerve growth factor immunoglobulin G specifically recognized nerve growth factor with no cross-reaction to recombinant brain-derived neurotrophic factor and neurotrophin-3 evaluated by an enzyme immunoassay. We quantified nerve growth factor content in each layer of the adult rat cerebral cortex and in each small piece (0.225 mg wet weight tissue) of the diencephalon, brainstem and cerebellum with a highly sensitive two-site enzyme immunoassay. Nerve growth factor content was unevenly distributed in the cerebral cortex (dense in layers II/III and V/VI and sparse in layers I and IV). Moderate to high levels of nerve growth factor were registered in the habenular nuclei, zona incerta, ventral tegmental area, substantia nigra, locus coeruleus, ventral cochlear nucleus, trapezoid body, lateral vestibular nucleus, cerebellar nuclei and paraflocculus. Immunohistochemically, the nerve growth factor-like immunoreactivity was found in the cell bodies, dendrites and axons of adult rat central neurons, not only in the cerebral cortex, hippocampus and basal forebrain, but also in the diencephalon, brainstem and cerebellum. The population of neurons with nerve growth factor-like immunoreactivity was limited, but unexpectedly widespread, and the density of these cells correlated well with the content determined by an enzyme immunoassay in the present and a previous study [Nishio T. et al. (1992) Expl Neurol. 116, 76–84]. The monoamine neurons, including dopaminergic, noradrenergic and serotonergic neurons, showed intense nerve growth factor-like immunoreactivity, indicating that the central monoaminergic neuronal system may also be involved in the nerve growth factor trophic system. To visualize nerve growth factor transported in the axons and to enhance the immunostaining in the nerve growth factor-producing cells, we injected colchicine, a potent inhibitor of microtubule polymerization and a blocker of axoplasmic transport, into the lateral ventricle of adult Wistar rat brain. Colchicine treatment enhanced the intensities of nerve growth factor-like immunoreactivity in the axons and cell bodies, especially in the axon hillocks and the proximal axons of the nerve growth factor-producing neurons. This observation may suggest the existence of an orthograde axonal transport system for nerve growth factor in the central neurons. These results suggest that nerve growth factor may exist in widespread populations of adult central neurons, including the monoaminergic neurons, and may exert a trophic effect on these neurons through not only a retrograde manner but also an orthograde manner of transport.

Retrograde axonal transport of the alpha-subunit of the GTP-binding protein GZ in mouse sciatic nerve: a potential pathway for signal transduction in neurons.

We have utilized antibodies against the alpha subunit of GZ in fluorescence immunohistochemistry to determine whether this GTP-binding protein can translocate along nerves by intra-axonal transport. After ligation of the mouse sciatic nerve we found an increase in GZ-like immunoreactivity on the proximal and distal side with time, suggesting that the alpha subunit undergoes orthograde axonal transport and also returns to the cell body by retrograde axonal transport in the sciatic nerve. Unlike the retrograde transport of Gi alpha, shown in a previous study to be present in most sciatic axons, GZ alpha only accumulated in a subpopulation of axons, suggesting that different G-proteins could convey information specific to neuronal subtypes. These results support our proposal that GZ may play a second messenger role in communicating information from the terminals back to cell bodies. Gi alpha and GZ alpha may be representative of relatively stable signalling molecules by which the signal from some neurotrophic molecules can be translocated from the neuron periphery to the cell body without the need for the retrograde transport of the neurotrophic factor itself.

Orthograde transport and release of insulin-like growth factor I from the inferior olive to the cerebellum.

Insulin-like growth factor I (IGF-I) and its receptor are expressed in functionally related areas of the rat brain such as the inferior olive and the cerebellar cortex. A marked decrease of IGF-I levels in cerebellum is found when inferior olive neurons are lesioned. In addition, Purkinje cells in the cerebellar cortex depend on this growth factor to survive and differentiate in vitro. Thus, we consider it possible that IGF-I forms part of a putative trophic circuitry encompassing the inferior olive and the cerebellar cortex and possibly other functionally connected areas. To test this hypothesis we have studied whether IGF-I may be taken up, transported, and released from the inferior olive to the cerebellum. We have found that 125I-IGF-I is taken up by inferior olive neurons in a receptor-mediated process and orthogradely transported to the cerebellum. Thus, radioactivity found in the cerebellar lobe contralateral to the injection site in the inferior olive was immunoprecipitated by an anti-IGF-I antibody, co-eluted with 125I-IGF-I in an HPLC column, and co-migrated with 125I-IGF-I in an SDS-urea polyacrylamide gel electrophoresis. Time-course studies indicated that orthograde axonal transport is relatively rapid since 30 min after the injection, radiolabeled IGF-I was already detected in the contralateral cerebellum. Furthermore, transport of IGF-I from the inferior olive is specific since when 125I-neurotensin was injected in the inferior olive or when 125I-IGF-I was injected in the pontine nucleus, no radioactivity was found in the contralateral cerebellum.(ABSTRACT TRUNCATED AT 250 WORDS)

Acoustic chiasm IV: Eight midbrain decussations of the auditory system in the cat

Conventional retrograde and orthograde axonal transport tract-tracing techniques were used in cats to explore the auditory decussations and commissures in the upper pons and midbrain. In all, 8 decussations differing either in origin or in contralateral termination were found. Three of the 8 decussations (from the dorsal nucleus of the lateral lemniscus to the contralateral dorsal nucleus of the lateral lemniscus, from the dorsal nucleus of the lateral lemniscus to the contralateral inferior colliculus, from the sagulum to the contralateral sagulum) reach their targets via the commissure of Probst. The remaining 5 decussations (from the inferior colliculus to the contralateral inferior colliculus or medial geniculate, from the intermediate nucleus of the lateral lemniscus to the contralateral medial geniculate, from the sagulum to the contralateral inferior colliculus or medial geniculate) reach their targets via the commissure of the inferior colliculus. The results also suggest that the commissure of Probst is not a general avenue for decussating auditory fibers of the lateral lemniscus but is instead a specific avenue only for fibers from the dorsal nucleus of the lateral lemniscus and sagulum. The results also show that, in the cat at least, the dorsal nucleus of the lateral lemniscus does not project beyond the inferior colliculus to either the superior colliculus or medial geniculate--the cells previously reported as doing so are probably those of the immediate neighbors of the dorsal nucleus, the intermediate nucleus of the lateral lemniscus and sagulum.

Organization of cortical afferent and efferent pathways in the white matter of the rat visual system

Fibers forming reciprocal connections between the dorsal lateral geniculate nucleus and primary visual cortex run in separate tracts in the white matter. The corticofugal fibers are organized into bundles which project through the coronal plane at an oblique angle. We have examined the organization of corticofugal fiber tracts within the white matter of the rat using cortical slices which encompassed the predicted trajectory of these fiber bundles. Field potentials were evoked in layer VI of visual cortex by focal stimulation of subcortical white matter using microbipolar electrodes. Two major responses were elicited: a short-latency and a longer-latency response. The short-latency response was elicited in the superficial strata of white matter with proximal stimulation sites and was obtained in deeper strata for more distant, lateral sites. The longer-latency response was associated with superficial strata in white matter at both proximal and distant stimulation sites. Based on the electrophysiological properties and the white matter location for eliciting these responses, it is likely that the short-latency response is due to antidromic activation of corticogeniculate fibers, whereas the longer-latency response probably arises from orthodromic activation of geniculocortical fibers. These findings provide an electrophysiological demonstration that cortical afferent and efferent pathways are segragated within the white matter and that they can be selectively activated by focal stimulation. The fact that the fiber bundle model successfully predicted the trajectory of corticofugal fibers provides additional support for this model of white matter organization.A double labeling technique which combined orthograde axonal transport and neuronal degeneration was used to examine the topographic arrangement of corticofugal fibers in the white matter. Corticofugal fibers are topographically organized in subcortical white matter in a manner which reflects the location of their cell bodies in cortex. Fibers arising from neurons situated laterally and rostrally in the cortex are superficial to those originating from more medial and caudal neurons.This topographic ordering of fibers in the white matter suggests that contact guidance may play an important role during axonogenesis in directing corticofugal axons to appropriate subcortical targets.

Fast axonal transport of labeled proteins in motoneurons of exercise-trained rats

In this study, the fast orthograde axonal transport of radiolabeled proteins was measured to determine the effects of endurance-running training on transport velocity and amounts of transported proteins in rat sciatic motoneurons. Female rats were subjected to a progressive running-training program for 10-12 wk. Twenty-four hours after the last training session, rats underwent right L4-L5 dorsal root ganglionectomy. The next day, 20 microCi of [3H]leucine was injected bilaterally in the vicinity of the motoneuronal cell bodies supplying the sciatic nerve, to study axonal transport parameters. Results showed that peak and average transport velocities of labeled proteins were significantly (P less than 0.05) increased by 22 and 29%, respectively, in the deafferented nerves of the runners as compared with controls. Moreover, the amount of total transported protein-bound radioactivity was increased in both left (40%) and right (37%) sciatic nerves of the runners. An exhaustive exercise session reduced (P less than 0.05) peak displacement (8%) and total transported protein-bound radioactivity (36%) in the sciatic nerves of control rats, whereas no changes were noticed in trained animals. The data suggest that chronic endurance running induces significant adaptations in the fast axonal transport of labeled proteins.

Oxygen-induced retinopathy in newborn rats: Orthograde axonal transport changes in optic pathways

The aim of this study was to evaluate axonal transport along the optic pathways of neonatal rats in which oxygen-induced retinopathy had been experimentally produced. One group of 116 rats was exposed to an FiO 2 of 80% for the first 5 days of life and then kept under room-air conditions for the following 10 days. A second group of 108 rats was maintained under room-air conditions for the first 15 days of life. On the 16th day of life, 1·5 μl of [ 35S]taurine was injected into the vitreous of the right eye of each animal and radioactivity was measured, at various intervals from the injection, in the right optic nerve, the chiasma and the left optic tract. Statistically significant reductions in axonal transport were observed in all three segments of the optic pathways of the oxygen-supplemented animals compared with the control group. The authors discuss various explanations for their findings and hypothesize that the alterations observed in this study may have significant and more or less long-term effects on the normal maturation of the visual system. kwoxygen-induced retinopathy; axonal transport; optic pathways; rats; oxygen; taurine

Dissociation of the inhibition of fast axonal transport by chlorimipramine from an effect on axonal microtubules

The effect of in vitro exposure of bullfrog spinal nerves to 0.2 mM chlorimipramine on the density of axonal microtubules was studied in an attempt to clarify the mechanism by which chlorimipramine inhibits fast axonal transport. A 17-h exposure to chlorimipramine reduced the density of microtubules in unmyelinated axons by only 18%; this microtubular loss does not reach the upper limit of the range of microtubule reduction associated with inhibition of fast axonal transport. A 23-h exposure to chlorimipramine, which had decreased microtubular density in unmyelinated axons by 40% in a previous study, did not decrease microtubular density in myelinated axons in the present study. These results rule out microtubular destruction as the mechanism responsible for inhibition of fast orthograde axonal transport by chlorimipramine, and greatly reduce the likelihood that microtubular destruction plays a significant role in the inhibition of fast retrograde transport by chlorimipramine.

Distribution and orthograde axonal transport of HRP in feline eyes following intravitreal injection: an ultrastructural study

The retina and optic nerve in the cat were examined by electron microscopy at five different time periods (1-, 3-, 8-, 16- and 24 hr) following the injection of horseradish peroxidase (HRP) into the vitreous cavity. Intra- and intercellular HRP was found in ganglion cells, Müller cells and horizontal cells even at 1 hr. Intra-axonal HRP in the retina and the retrolaminar optic nerve was seen most frequently at 3- and 8 hr. At 16- and 24 hr, many phagocytes containing HRP were noted in the nerve-fibre layer; subsequently HRP activity decreased throughout the retina. On the other hand, HRP activity in the intracanalicular optic nerve was significantly increased at 16 hr. Intra-axonal HRP appeared to be morphologically distributed in two forms: (i) HRP granules bound by a single-unit membrane mostly 0.1-0.4 micron in diameter; (ii) granules less than 0.05 micron which were occasionally hard to distinguish from artefacts such as stain deposit. The rate of orthograde axonal transport of HRP granules larger than 0.05 micron was examined on the basis of the frequency of HRP granules appearing in the retrolaminar and intracanalicular parts of the optic nerves. Though the rate seemed to be widely variable, the major velocity was calculated to be 45 mm per day, which is equal to intermediate axonal flow rates.

Optic nerve head axonal transport in rabbits with hereditary glaucoma

Rabbits with hereditary glaucoma develop ocular changes that resemble human congenital glaucoma and buphthalmia. The inheritance is autosomal recessive (bu). Previous research was performed primarily on albino bu/bu rabbits that were unhealthy and bred poorly. We have bred pigmented bu/bu rabbits to determine if this would improve hardiness and provide a better model for the disease in humans. First-generation offspring from matings of bu/bu albino with bu/bu pigmented rabbits were all affected, indicating that the bu gene is found at the same locus in both strains. The pigmented bu/bu offspring had a high degree of mortality, as reported previously for albino bu/bu rabbits. Newborn bu/bu rabbits initially had normal intraocular pressure (IOP; 15-23 mmHg); after 1- to 3 months, the IOP increased to 26-48 mmHg. The eyes became buphthalmic and the IOP returned to normal or sub-normal levels after 6-10 months. Since the lamina cribrosa is absent or poorly formed in the rabbit optic nerve head (ONH), this model was used to test the role of mechanical factors in the etiology of ONH pathology caused by increased IOP. Orthograde axonal transport was evaluated in both eyes from eight normal and 24 bu/bu rabbits of different ages, using intravitreal injections of [3H]leucine to mark orthograde axonal transport, followed by light- and electron-microscopic radioautography of the ONHs and superior colliculi. Normal rabbits of all ages showed no blockage of axonal transport in the ONH. All optic axons from young bu/bu rabbits with normal IOP and most axons from older buphthalmic rabbits that previously had elevated IOP were normal morphologically. Small zones of transport blockage occurred in bu/bu eyes while IOP was elevated; most affected axons lay immediately adjacent to ONH connective tissue beams that radiate outward from the central retinal vessels to the optic-nerve sheath. Thus, the rabbit, which lacks a true lamina cribrosa, does not show marked blockage of axonal transport as occurs in the LS of the monkey and cat ONH when IOP is elevated acutely. This anatomic difference appears to be protective against axonal damage, since bu/bu rabbits with chronic IOP elevation did not show significant loss of optic axons. These results are consistent with the proposed 'mechanical' theory of ONH damage resulting from increased IOP. Electron-microscopic radioautography revealed that chronically elevated IOP in bu/bu rabbits, which caused small foci of blocked ONH axonal transport against ONH beams, also caused degeneration of a few optic nerve terminals in the superior colliculi as the disease progressed.(ABSTRACT TRUNCATED AT 400 WORDS)

Ornithine decar☐ylase activity in dorsal root ganglia of regenerating frog sciatic nerve

Ornithine decar☐ylase (ODC) activity was studied in dorsal root ganglia (DRG) of regenerating frog sciatic nerve. There was a significant increase in activity two days after a crush lesion of the nerve 2.5 cm distal to the DRG. The increase reached a maximum after 7 days, then declined but remained above control levels for at least 9 days. An endoneural injection of vinblastine, a potent inhibitor of retrograde and orthograde axonal transport, between the DRG and the crush inhibited the increase in ODC. In contrast, injections of vinblastine into undamaged nerves failed to affect ODC. The increase in ODC and also the regenerative properties of the nerve could be prevented by daily i.p. injections of α-difluoromethyl ornithine. We suggest that a signal is formed at the site of injury in the sciatic nerve. This signal is conveyed to the DRG by retrograde axonal transport where it initiates the events leading to an increase in ODC. This increase appears to be necessary for the regeneration of sensory fibers in the frog sciatic nerve.

Studies of aldose reductase using neuronal cell culture and ligated rat sciatic nerve

Sorbinil (CP 45,634), a potent aldose reductase (AR) inhibitor, has the ability to normalize both sorbitol levels and functional parameters such as orthograde axonal transport and motor nerve conduction velocity in peripheral nerves of diabetic rats, which implicates flux through the polyol pathway in the pathophysiology of diabetic neuropathy. In order to understand more fully the role of this enzyme, it is important to determine the major cellular location of AR in peripheral nerve. Experiments were designed that have taken advantage of the observation that peripheral nerve axons degenerate distal to an injury site, while Schwann cells remain viable. One sciatic nerve in each experimental rat was chronically ligated (up to 6 weeks) before inducing diabetes by an intravenous (iv) injection of streptozotocin (STZ; 65 mg/kg). Two days after the STZ injection, both sciatic nerves were removed from each animal, and the ligated nerve was divided into proximal (Schwann cells and axons) and distal (Schwann cells only) portions before being processed for sorbitol determinations. The intact nerves and the proximal protion of the ligated nerves (having both Schwann cells and axons) retained the ability to accumulate sorbitol after STZ injection, while the distal portion (having Schwann cells only) lost this capacity 4 days after ligation. This lack of ability to accumulate sorbitol was not due to failure of the substrate (glucose) to reach the distal nerve segment. Additionally, homogenates of whole nerves and of proximal portions of ligated nerves were able to form sorbitol from glucose in the presence of NADPH while homogenates of distal portions of ligated nerves had lost approximately 85% of this activity. In addition, when glyceraldehyde was used as the substrate, the distal segment homogenates lost 70% of the glycerol-synthesizing activity seen in whole nerve homogenates. These data contrast with previous reports that showed no loss of AR activity in distal ends of transected rabbit sciatic nerve. The fact the neuronal tissue has AR-like activity is demonstrated in studies utilizing both primary cultures of rat sympathetic neurons and the PC12 pheochromocytoma cell line; both of these accumulate sorbitol when grown in high-glucose medium. This accumulation is inhibited in a dose-dependent manner by the inclusion of sorbinil. It has been found that the distal portions of ligated nerves neither accumulate sorbitol nor retain the ability to make sorbitol under conditions where both whole nerves and proximal segments of ligated nerves do accumulate and synthesize this sugar. These findings are consistent with at least two interpretations that are not mutually exclusive. The first is that sorbitol-synthesizing activity (AR) may be located in axons rather than in Schwann cells, as previously reported. Alternatively, the sorbitol-synthesizing activity may be a differentiated function of Schwann cells, which is lost as the Schwann cells de-differentiate after ligation. The former interpretation of the data is supported by the observation that primary cultures of rat sympathetic neurons can make sorbitol. This interpretation lends support to the hypothesis that peripheral diabetic neuropathy is a primary dysfunction of axons. Further experimentation will attempt to distinguish between these two possibilities.