Topographic disorganization of the optic tracts following long-term optic nerve regeneration: a quantitative image analysis study

Experiments were designed to find the degree to which regenerated optic axons occupied their previous locations in the optic tracts. Following optic nerve crush and regeneration, either the dorsal, ventral, peripheral, temporal, or nasal part of the retina was ablated. The axons of the remaining retinal ganglion cells (RGCs) were labeled with cobalt. Density of the regenerated dorsal and ventral axons in the dorsal vs. ventral optic tracts was determined digitally. In addition, we determined the density of temporal and nasal axons in the temporal vs. nasal compartments of each optic tract and the density of central axons in the central vs. peripheral compartments of both optic tracts. Regenerated axons were not distributed randomly in the optic tracts. Instead, they were slightly but, significantly biased toward growing through the tract or compartment that they had occupied previously. Still, the pathway specificity exhibited by the regenerated axons was closer to random than it was to the pathway specificity seen in normal animals. Dorsal, ventral, and central RGC axons were significantly better localized to their correct tract or compartment than were temporal or nasal RGC axons. Also, over time, dorsal and ventral axons tended to disappear from incorrectly chosen optic tracts. The slight bias toward choosing the appropriate optic tract or optic tract compartment may be enough to account for the topographic specificity of the regenerated retinotectal projection. Near-randomness of the axonal positions in the tracts argues against the presence of any specific guidance cues in the optic tracts of adult animals. Axonal density was highest in the correct compartment and diminished progressively with increasing distance into the incorrect compartment. Such a gradient of axonal density suggests that regenerating axons "drift" away from their previous positions in the optic pathways.     

Astroglial-axonal interrelationship during regeneration of the optic nerve in goldfish. A freeze-fracture study

The cytoplasmic membranes of astrocytes in the regenerating optic nerve of the goldfish were investigated by means of freeze-fracture technique. In contrast to avian and mammalian astrocytes the astrocytes in the fish white matter do not reveal the characteristic orthogonal particle assemblies. Whereas astrocytes hitherto were believed to be interconnected mainly by gap junctions, astrocytes in the optic nerve of the goldfish are additionally interconnected by prominent desmosomes and large tight junctions. The tight junctional strands are frequently intercalated by gap junctions. Astroglial membrane domains, which are directly juxtaposed to untreated myelinated optic fibers, are longitudinally deformed. The distance of these longitudinal grooves corresponds to the caliber of fibers associated with them. The intramembranous particles in these membranous domains are distributed randomly. During optic nerve regeneration, membranes of astrocytes associated with regenerating, still unmyelinated fibers are either longitudinally deformed or smooth. If deformed according to the axonal course the membrane shows a non-random distribution of intramembranous particles; if smooth, the membrane shows a random distribution of its particles. The non-random particle distribution in the astrocytic P-face is characterized by an impoverishment of particles along the concave groove and an enrichment of particles along the convexe bulge which corresponds to the wedge between two associated axons. This phenomenon is restricted to the regeneration period and disappears some weeks after recovery of vision. Then the astrocytic membranes which are associated to regenerated fibers are undulated nevertheless revealing a random particle distribution. It is suggested, that these findings reflect a glial-neuronal interrelationship characterized by the neuron as inductor and the glial cell as responder to axonal signals. The astroglial response to axonal signals would include the formation of attractive glial channels. The working hypothesis is proposed that the presence of orthogonal particle assemblies could contribute to a disturbed astroglial response in the mammalian central nervous system.     

Stages of axonal regeneration following optic nerve crush in goldfish: contrasting effects of conditioning nerve lesions and intraocular acetoxycycloheximide injections

The progress of axonal outgrowth after a crush lesion of the goldfish optic nerve can be determined by examining longitudinal silver-stained sections at selected intervals. The outgrowth of leading axons proceeded at 0.46 mm/day after an initial delay of 4.2 days. Outgrowth can be rapidly characterized by differentiating among a series of qualitatively different stages. In the sprouting (S) phase of regeneration, stage S1 is defined by the presence of isolated axonal sprouts reaching into the crush zone, and stage S2 by bundles of sprouts in the crush zone. In the elongation (E) phase of regeneration, stage E1 is defined by bundles that bridge the crush zone, stage E2 by bundles that reach the optic chiasm, and stage E3 by bundles that reach the level of the hypothalamus. During normal regeneration, stage E2 was attained 7-9 days after the crush (testing lesion), and stage E3 at 11 days. However, if the testing lesion had been preceded by a similar (conditioning) lesion 2 weeks earlier, stage E2 was reached at 3 days and stage E3 at 5 days. Conversely, when a protein synthesis inhibitor (acetoxycycloheximide) was injected into the right eye daily from the 5th through 9th day after a testing lesion, the injected side lagged 1-2 stages behind the contralateral control side in nerves examined on the 10th day.     

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.     

Immunological evidence that the neural adhesion molecule L1 is expressed in fish brain and optic nerve: possible association with optic nerve regeneration

In the mammalian peripheral nervous system (PNS), expression of the neural adhesion molecule L1 on Schwann cells and neurons has been correlated with axonal growth during development and regeneration. The present study was undertaken to examine whether a similar correlation exists between a lesion-induced increase of L1 expression and regenerative capacity in the central nervous system (CNS). The fish optic nerve was used as a model for a succesfully regenerating region of the CNS. Immunochemical and immunohistological experiments carried out with immunoaffinity purified polyclonal antibodies, generated against L1 from mouse brain, showed that carp optic nerve and brain, but not liver, contained L1 immunoreactivity. Western blot analysis of brain tissue yielded one distinct band at 200 kDa, while a double band at 200 kDa and two low-molecular weight bands at 120 and 100 kDa, possibly degradation products, were seen in the optic nerve. Immunohistological examination of normal optic nerves revealed L1 immunoreactivity, predominantly associated with connective tissue boundaries of nerve fascicles and with blood vessels, as well as inside axonal fascicles. L1 immunoreactivity was increased by 25%, 8 days after crushing of the optic nerve, as determined by radioimmunoassay on a nerve segment distal to the site of injury and compared with untreated control nerves. Increased levels of L1 were also seen by immunohistology and found to be predominantly associated, as in the normal nerve, with connective tissue boundaries and blood vessels. These observations suggest that a lesion-induced increase in L1 expression in the fish optic nerve is associated with axonal regrowth in the CNS.     

An electrophysiological study of early retinotectal projection patterns during regeneration following optic nerve crush inside the cranium in Hyla moorei

The sequence of regeneration following intracranial optic nerve crush has been studied using electrophysological visual mapping in the frog Hyla moorei. Compared to our earlier series⁹ with extracranial crush, the time course was slower and the intermediate projections more disorganized. It is suggested that the apparent discrepancy between the early patterns of regeneration in Xenopus⁴ and Rana^5,6 compared to Hyla⁹, fish⁸ and newt², is not due to species differences but to the location of the lesion site.     

Altered synaptic organization in facial nucleus following facial nerve regeneration: An electrophysiological study in man

Seven patients with postfacial palsy contracture and mass contractions were investigated electrophysiologically. In 3 patients the early blink reflex showed an unusually high amplitude, which can be attributed to enhanced excitability of facial motor neurons. In 5 patients the early blink reflex had acquired a crossed character. It is assumed that changes in organization of the facial nucleus contribute to the altered function of reinnervated facial muscles.     

Peripheral nerve grafts to the frog optic tectum: a morphological study of foreign axon regeneration in the central nervous system

The proximal stump of a transected mandibular nerve was grafted onto the rostrodorsal surface of the optic tectum in adult Rana pipiens to investigate the morphologic characteristics of nonspecific axonal regeneration in a highly organized region of central nervous system (CNS). Within the first 3 weeks postgraft surgery (WPS), the nerve-tectum interface became firmly established. Concomitant with this was an invasion of the host tectum by a small number of fine "pioneerlike" axons from the nerve. By 6 WPS there developed a concerted instreaming of a large number of peripheral fibers. Once within the CNS, the foreign axons distributed themselves throughout the rostrocaudal extent of the tectum, but primarily its dorsal aspect within superficial layers 8 and 9. Presence of intact optic fibers at the time of mandibular fiber invasion served somewhat to restrict the regenerating aberrant axons in their course through layer 9. This restriction could be avoided by removal of the optic input either before or during peripheral ingrowth. However, once peripheral fibers had entered and established themselves in the host environment, no subsequent manipulation of the retinotectal projection had any effect. The aberrant growth pattern, which appeared remarkably stable after 6 WPS, consisted of a plexus of medium- and fine-caliber peripheral axons. Many of these fibers had numerous branches and "en passant" varicosities, the latter encompassing a variety of shapes and sizes. Terminal swellings and arborizations were also found. When comparing the regeneration of optic and mandibular nerve fibers in the tectum, two distinctions were made. Whereas optic axons revealed a fascicular and layered organization, mandibular axons showed a highly segregated and disordered growth pattern. These characteristic differences were maintained even when the two fiber systems were allowed to coregenerate into the same target tectum. Thus, each of the two groups of axons interacts with the tectal substrate in a distinct manner, apparently independent of the other.     

A collagen-based nerve guide conduit for peripheral nerve repair: an electrophysiological study of nerve regeneration in rodents and nonhuman primates

When a peripheral nerve is severed and left untreated, the most likely result is the formation of an endbulb neuroma; this tangled mass of disorganized nerve fibers blocks functional recovery following nerve injury. Although there are several different approaches for promoting nerve repair, which have been greatly refined over recent years, the clinical results of peripheral nerve repair remain very disappointing. In this paper we compare the results of a collagen nerve guide conduit to the more standard clinical procedure of nerve autografting to promote repair of transected peripheral nerves in rats and nonhuman primates. In rats, we tested recovery from sciatic nerve transection and repair by 1) direct microsurgical suture, 2) 4 mm autograft, or 3) entubulation repair with collagen-based nerve guide conduits. Evoked muscle action potentials (MAP) were recorded from the gastrocnemius muscle at 4 and 12 weeks following sciatic nerve transection. At 4 weeks the repair group of direct suture demonstrated a significantly greater MAP, compared to the other surgical repair groups. However, at 12 weeks all four surgical repair groups displayed similar levels of recovery of the motor response. In six adult male Macaca fascicularis monkeys the median nerve was transected 2 cm above the wrist and repaired by either a 4 mm nerve autograft or a collagen-based nerve guide conduit leaving a 4 mm gap between nerve ends. Serial studies of motor and sensory fibers were performed by recording the evoked MAP from the abductor pollicis brevis muscle (APB) and the sensory action potential (SAP) evoked by stimulation of digital nerves (digit II), respectively, up to 760 days following surgery. Evoked muscle responses returned to normal baseline levels in all cases. Statistical analysis of the motor responses, as judged by the slope of the recovery curves, indicated a significantly more rapid rate of recovery for the nerve guide repair group. The final level of recovery of the MAP amplitudes was not significantly different between the groups. In contrast, the SAP amplitude only recovered to the low normal range and there were no statistically significant differences between the two groups in terms of sensory recovery rates. The rodent and primate studies suggest that in terms of recovery of physiological responses from target muscle and sensory nerves, entubulation repair of peripheral nerves with a collagen-based nerve guide conduit over a short nerve gap (4 mm) is as effective as a standard nerve autograft.(ABSTRACT TRUNCATED AT 400 WORDS)     

Analysis of axonal regeneration through the silicone regeneration chamber: A retrograde tracing study in the rabbit facial nerve

Transected facial nerve buccal branches in the adult rabbit were sutured to a silicone growth chamber and regeneration was observed at 3, 5, and 7 weeks postoperation. Using the retrograde axonal transport of horseradish peroxidase technique the soma in the facial motor nucleus were counted and the number was correlated with the number of axons found in the mid-cross section of the regenerating buccal branch and with the number of axons in the original nerve. The somatotopic reorganization in the facial motor nucleus was examined. The mean number of facial motoneuron soma labeled with HRP in the control was 68.0% (+/- 18.6, SE) of the axons counted at periphery. In the regenerating nerves, the labeled soma represented 2.4% of the preoperative controls after 3 weeks and rose to 8.6 and 43.9% at 5 and 7 weeks, respectively. At 3 weeks postoperative time, four of six regenerating nerves did not contain any myelinated axons at the center cross section. The ratios of labeled soma to the regenerating myelinated axons counted at the center cross section at 5- and 7-week time points were 42.2 and 61.1%, respectively. The location of the soma found in the early regeneration phase was similar to the normal distribution except in the dorsal subnucleus. After 7 weeks the proportion of labeled soma in the intermediate subnucleus declined but the general pattern replicated the distribution found in normal control preparations.     

Recovery of vision in fish after optic nerve crush: A behavioral and electrophysiological study

Recovery of the visual field was studied during regeneration of the retinotectal projection in bluegill sunfish (Lepomis macrochirus) using a behavioral perimetry technique. Fish were trained to orient ballistically to brief light flashes along the horizontal meridian. After crushing the right optic nerve intraorbitally, orienting to stimuli in the right visual field in different fish reappeared between 32 and 56 days postcrush. On average, recovery of the field center (30 to 150°) occurred in 3 days, the most temporal 30° requiring a further 7 days. Orientation accuracy, measured by the mean absolute error angle, returned to normal 10 to 15 days from the start of recovery. Perimetry was terminated at different times to 152 days postcrush, and the retinotectal projection was mapped electrophysiologically using an automated procedure to characterize multiunit receptive fields (MURFs). At early stages of visual recovery, MURFs associated with strong multiunit discharges were found in the same field regions responsive in behavioral perimetry. The 10- to 15-day recovery of normal orienting accuracy was associated with an increase in the amplitude of multiunit activity, attaining a plateau of 75% of normal. There was also evidence of a long-term decline in MURF area. Early maps showed a roughly normal visutopic order, although abnormalities in MURF size and shape persisted to as much as 152 days postcrush.     

Decreased axonal calibres without axonal loss in optic nerve following chronic alcohol feeding in adult rats: a morphometric study

Summary The effects of chronic ethanol exposure on number and calibres of optic nerve axons (and number of retinal ganglion cells) were investigated in a rat model. Male Sprague-Dawley rats were fed a liquid, ethanol-containing diet for 5, 10 and 17 weeks with littermates given isocaloric amounts of ethanol-free diet serving as controls. After fixation by perfusion, the optic nerves were imbedded in epoxy resin and sectioned for electron microscopy. Systematic random sampling was made from a cross-shaped area over the nerve. Axons within a counting frame were counted and morphometrically categorized with regard to mean diameter and the total number of axons estimated from number per area and the cross-sectional area of the nerve, which was measured using a digitizer table. According to non-parametric statistical analysis, ethanol exposure resulted in a significant reduction in mean cross-sectional area of the optic nerve and in mean axonal calibre but not in total axonal number in the ethanol-treated rats but there was no significant effect of duration of the exposure. The mean cross-sectional area of the nerve was reduced by 9%, 10% and 18% after 5, 10 and 17 weeks of exposure, respectively. The reduction in cross-sectional area appeared to be related to a proportional reduction in axonal and myelin area fractions. The findings indicate that chronic ethanol exposure results in decreased axonal calibres without axonal loss. This also implies that there is no reduction in the number of retinal ganglion cells.Supported by the Bank of Sweden Tercentenary Foundation (Proj. No. 86/80), the Swedish Medical Research Council (Proj. No. 07121) and Swedish Alcohol Monopoly Foundation for Alcohol Research (89/11)     

Transient up-regulation of retinal EphA3 and EphA5, but not ephrin-A2, coincides with re-establishment of a topographic map during optic nerve regeneration in goldfish

Eph tyrosine kinase receptors and their ligands, the ephrins, play a key role in the establishment of retinotectal topography during development. Tectal up-regulation of ephrin-A2 in goldfish, coincident with the reestablishment of a retinotectal map, suggests a similar role during optic nerve regeneration. Here we report a complementary study of EphA3, EphA5 and ephrin-A2 expression in the retina. EphA3 and EphA5 are transiently up-regulated as ascending naso-temporal gradients, whereas ephrin-A2 remains uniform. The expression profiles differ from those in developing chick and mouse, suggesting that different combinations of retinal Eph receptors and ligands can generate topographic guidance information.     

A study of the effects of isaxonine on vincristine-induced peripheral neuropathy in man and regeneration following peripheral nerve crush in the rat.

Administration of isaxonine (6 mg/kg powdered diet) had no effect on regeneration following sciatic nerve crush in the rat. In 10 patients undergoing treatment with vincristine (1.4 mg/m2 twice monthly) development of peripheral neuropathy was quantitated by neurological symptoms, signs and electrophysiological tests. Five also received isaxonine (1.5 g daily). All patients developed evidence of neuropathy, but in none was it severe. The three lowest disability scores were obtained in isaxonine treated patients, but the highest score was also in an isaxonine treated patient. The equivocal findings in this small study could not be amplified because the drug was withdrawn from clinical use on account of its hepatotoxicity.     

A quantitative histochemical study of early cellular events associated with destruction of myelinated axons in rat optic nerve

Microchemical techniques correlated with histologic observations in adult rat optic nerve were used to study the response of neuroglia to progressive membranous destruction of myelinated axons between 1 and 200 days after surgical removal of the eye. Cell enumeration was accomplished by combining DNA assays with differential cell counts in electron microscopic montages previously reported by others. During the first week after nerve transection, ganglioside sialic acid and proteolipid proteins decreased by 57% and 28%, respectively; these changes preceded the subsequent reduction of cerebrosides and cholesterol and light microscopic evidence of myelin destruction. This suggests that degeneration of axon cylinders and degradation of the protein core of myelin may be initiating events which induce instability in the lipid structure and subsequent fragmentation of the sheath. Transformation of astrocytes into undifferentiated glia with high ribosomal content also occurs during the initial phase of axonal degeneration, yielding a new line of cells capable of engulfing and digesting membranous debris. The slow clearance of degenerating fibers at about one-third the rate reported for peripheral nerve may be explained by the lack of infiltration of macrophages from blood and the relatively limited capacity of undifferentiated glia for phagocytosis.     

Compensatory strategies following visual search training in patients with homonymous hemianopia: an eye movement study

A total of 29 patients with homonymous visual field defects without neglect practised visual search in 20 daily sessions, over a period of 4 weeks. Patients searched for a single randomly positioned target amongst distractors displayed for 3 s. After training patients demonstrated significantly shorter reaction times for search stimuli (Pambakian et al. in J Neurol Neurosurg Psychiatry 75:1443–1448, 2004). In this study, patients achieved improved search efficiency after training by altering their oculomotor behaviour in the following ways: (1) patients directed a higher proportion of fixations into the hemispace containing the target, (2) patients were quicker to saccade into the hemifield containing the target if the initial saccade had been made into the opposite hemifield, (3) patients made fewer transitions from one hemifield to another before locating the target, (4) patients made a larger initial saccade, although the direction of the initial saccade did not change as a result of training, (5) patients acquired a larger visual lobe in their blind hemifield after training. Patients also required fewer saccades to locate the target after training reflecting improved search efficiency. All these changes were confined to the training period and maintained at follow-up. Taken together these results suggest that visual training facilitates the development of specific compensatory eye movement strategies in patients with homonymous visual field defects.     

Degeneration and regeneration in rabbit peripheral nerve with long-term nerve cuff electrode implant: a stereological study of myelinated and unmyelinated axons

Selective and dynamically co-ordinated functional electrical stimulation (FES) of paralysed/paretic limbs in upper motor neuron lesioned people depends on optimal contact at the neural interface. Implanted nerve cuff electrodes may form a stable electrical neural interface, but may also inflict nerve damage. In this study the immediate and long-term effects of cuff implantation on the number and sizes of myelinated and unmyelinated axons have been evaluated with unbiased stereological techniques. Cuff electrodes were implanted in rabbit tibial nerves just below the knee joint, and the stereological analyses were carried out 2 weeks and 16 months after implantation. Myelinated axons were analysed at standardised levels proximal to, underneath, and distal to the cuff; unmyelinated axons underneath the cuff. A 27% loss of myelinated axons was found underneath and distal to the nerve cuff 2 weeks post surgery. All axonal sizes were equally lost except for the very smallest. At 16 months post surgery the number of myelinated axons was restored to control values at both levels. Except for the presence of regenerative sprouts at 2 weeks post surgery, no changes in the number or sizes of unmyelinated axons were revealed at either 2 weeks or 16 months post surgery. Our study demonstrates that implanted cuff electrodes may cause an initial loss of myelinated axons but with subsequent regeneration. Received: 19 September 1997 / Revised: 27 February 1998 / Revised, accepted: 31 March 1998