Axonal regeneration of retinal ganglion cells after optic nerve pre-lesions and attachment of normal or pre-degenerated peripheral nerve grafts

Axonal regeneration of retinal ganglion cells (RGCs) into a normal or pre-degenerated peripheral nerve graft after an optic nerve pre-lesion was investigated. A pre-lesion performed 1-2 weeks before a second lesion has been shown to enhance axonal regeneration in peripheral nerves (PN) but not in optic nerves (ON) in mammals. The lack of such a beneficial pre-lesion effect may be due to the long delay (1-6 weeks) between the two lesions since RGCs and their axons degenerate rapidly 1-2 weeks following axotomy in adult rodents. The present study examined the effects of the proximal and distal ON pre-lesions with a shortened delay (0-8 days) on axonal regeneration of RGCs through a normal or pre-degenerated PN graft. The ON of adult hamsters was transected intraorbitallv at 2 mm. (proximal lesion) or intracranially at 7 mm (distal lesion) from the optic disc. The pre-lesioned ON was re-transected at 0.5 mm from the disc after 0, 1, 2, 4, or 8 days and a normal or a pre-degenerated PN graft was attached onto the ocular stump. The number of RGCs regenerating their injured axons into the PN graft was estimated by retrograde labeling with FluoroGold 4 weeks after grafting. The number of regenerating RGCs decreased significantly when the delay-time increased in animals with both the ON pre-lesions (proximal or distal) compared to control animals without an ON pre-lesion. The proximal ON pre-lesion significantly reduced the number of regenerating RGCs after a delay of 8 days in comparison with the distal lesion. However, this adverse effect can be overcome, to some degree, by a pre-degenerated PN graft applied 2, 4, or 8 days after the distal ON pre-lesion enhanced more RGCs to regenerate than the normal PN graft. Thus, in order to obtain the highest number of regenerating RGCs, a pre-degenerated PN should be grafted immediately after an ON lesion.

Cryosections of pre-irradiated adult rat spinal cord tissue support axonal regeneration in vitro

Neonatal X-irradiation of central nervous system (CNS) tissue markedly reduces the glial population in the irradiated area. Previous in vivo studies have demonstrated regenerative success of adult dorsal root ganglion (DRG) neurons into the neonatally-irradiated spinal cord. The present study was undertaken to determine whether these results could be replicated in an in vitro environment. The lumbosacral spinal cord of anaesthetised Wistar rat pups, aged between 1 and 5 days, was subjected to a single dose (40 Gray) of X-irradiation. A sham-irradiated group acted as controls. Rats were allowed to reach adulthood before being killed. Their lumbosacral spinal cords were dissected out and processed for sectioning in a cryostat. Cryosections (10 mum-thick) of the spinal cord tissue were picked up on sterile glass coverslips and used as substrates for culturing dissociated adult DRG neurons. After an appropriate incubation period, cultures were fixed in 2% paraformaldehyde and immunolabelled to visualise both the spinal cord substrate using anti-glial fibrillary acidic protein (GFAP) and the growing DRG neurons using anti-growth associated protein (GAP-43). Successful growth of DRG neurites was observed on irradiated, but not on non-irradiated, sections of spinal cord. Thus, neonatal X-irradiation of spinal cord tissue appears to alter its environment such that it can later support, rather than inhibit, axonal regeneration. It is suggested that this alteration may be due, at least in part, to depletion in the number of and/or a change in the characteristics of the glial cells. (C) 2000 ISDN. Published by Elsevier Science Ltd. All rights reserved.

Transport of endosomal early antigen I in the rat sciatic nerve and location in cultured neurons

Early endosomal antigen I (EEAI) is known to be a marker of early endosomes and in cultured hippocampal neurons it preferentially localizes to the dendritic but not the axonal compartment. We show in cultured dorsal root ganglia and superior cervical ganglia neurons that EEAI localizes to the cell bodies and the neurites of both sensory and sympathetic neurons. We then show in vivo using a ligated rat sciatic nerve that EEAI significantly accumulates on the proximal side and not on the distal side of the ligation. This suggests that EEAI is transported in the anterograde direction in axons either as part of the homeostatic process or to the nerve ligation site in response to nerve injury. NeuroReport 12:281-284 (C) 2001 Lippincott Williams & Wilkins.

Longitudinal excursion and strain in the median nerve during novel nerve gliding exercises for carpal tunnel syndrome

Nerve and tendon gliding exercises are advocated in the conservative and postoperative management of carpal tunnel syndrome (CTS). However, traditionally advocated exercises elongate the nerve bedding substantially, which may induce a potentially deleterious strain in the median nerve with the risk of symptom exacerbation in some patients and reduced benefits from nerve gliding. This study aimed to evaluate various nerve gliding exercises, including novel techniques that aim to slide the nerve through the carpal tunnel while minimizing strain (sliding techniques). With these sliding techniques, it is assumed that an increase in nerve strain due to nerve bed elongation at one joint (e.g., wrist extension) is simultaneously counterbalanced by a decrease in nerve bed length at an adjacent joint (e.g., elbow flexion). Excursion and strain in the median nerve at the wrist were measured with a digital calliper and miniature strain gauge in six human cadavers during six mobilization techniques. The sliding technique resulted in an excursion of 12.4 mm, which was 30% larger than any other technique (p 0.0002). Strain also differed between techniques (p 0.00001), with minimal peak values for the sliding technique. Nerve gliding associated with wrist movements can be considerably increased and nerve strain substantially reduced by simultaneously moving neighboring joints. These novel nerve sliding techniques are biologically plausible exercises for CTS that deserve further clinical evaluation. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 25:972-980, 2007

The expression of messenger RNAs coding for growth factors, their receptors, and eph-class receptor tyrosine kinases in normal and ototoxically damaged chick cochleae

Messenger RNAs coding for growth factors and receptor tyrosine kinases were measured by quantitative competitive and by semi-quantitative reverse-transcription polymerase chain reaction in whole and dissected chick inner ears. The fibroblast growth factor (FGF) receptor 1 chick embryonic kinase (CEK) 1 was expressed in all structures examined (otocyst, hatchling whole cochlea, cochlear nerve ganglion, and cochlear and vestibular sensory epithelia), although slightly more heavily in the otocyst. The related fibroblast growth factor receptors CEK 2 and 3 were preferentially expressed in the nerve ganglion and in the vestibular sensory epithelium, respectively. FGF 1 mRNA was low in early development, increasing to mature levels at around embryonic age 11 days, while FGF2, mRNA was expressed at constant levels at all ages. In response to ototoxic damage, FGF1 mRNA levels were increased in the early damaged cochlear sensory epithelium. Immunohistochemistry for CEK1 showed that normal hair cells expressed the receptor heavily on the hair cell stereocilia, while with early damage, CEK1 came to be expressed heavily on the apical surfaces of the supporting cells. In normal chicks, the CEK4 and CEK8 eph-class receptor tyrosine kinases were expressed relatively heavily by the cochlear nerve ganglion, and CEK10 was expressed relatively heavily by the cochlear hair cell sensory epithelium. The results suggest that the FGF system may be involved in the response of the cochlear epithelium to ototoxic damage. The eph-class receptor tyrosine kinase CEK10 may be involved in cell interactions in the cochlear sensory epithelium, while CEK4 and CEK8 may play a role in the cochlear innervation.

Kinetic analysis of vascular marker distribution in perfused rat livers after regeneration following partial hepatectomy

Background/Aims: Liver clearance models are based on information (or assumptions) on solute distribution kinetics within the microvasculatory system, The aim was to study albumin distribution kinetics in regenerated livers and in livers of normal adult rats, Methods: A novel mathematical model was used to evaluate the distribution space and the transit time dispersion of albumin in livers following regeneration after a two-thirds hepatectomy compared to livers of normal adult rats. Outflow curves of albumin measured after bolus injection in single-pass perfused rat livers were analyzed by correcting for the influence of catheters and fitting a long-tailed function to the data. Results: The curves were well described by the proposed model. The distribution volume and the transit time dispersion of albumin observed in the partial hepatectomy group were not significantly different from livers of normal adult rats. Conclusions: These findings suggest that the distribution space and the transit time dispersion of albumin (CV2) is relatively constant irrespective of the presence of rapid and extensive repair. This invariance of CV2 implies, as a first approximation, a similar degree of intrasinusoidal mixing, The finding that a sum of two (instead of one) inverse Gaussian densities is an appropriate empirical function to describe the outflow curve of vascular indicators has consequences for an improved prediction of hepatic solute extraction.

Enhanced downregulation of the p75 nerve growth factor receptor by cholesteryl and bis-cholesteryl antisense oligonucleotides

The effects of conjugating cholesterol to either or both ends of a phosphorothioate (PS) oligonucleotide were analyzed in terms of cellular uptake and antisense efficacy. The oligo sequence was directed against the p75 nerve growth factor receptor (p75), and was tested in differentiated PC12 cells, which express high levels of this protein. The addition of a single cholesteryl group to the 5'-end significantly increased cellular uptake and improved p75 mRNA downregulation compared with the unmodified PS oligo, However, only a minor degree of downregulation of p75 protein was obtained with 5' cholesteryl oligos, Three different linkers was used to attach the 5' cholesteryl group but were found not to have any impact on efficacy. Addition of a single cholesteryl group to the 3'-end led to greater p75 mRNA downregulation (31%) and p75 protein downregulation (28%) than occurred with the 5' cholesteryl oligos. The biggest improvement in antisense efficacy, both at the mRNA and protein levels, was obtained from the conjugation of cholesterol to both ends of the oligo. One of the bis-cholesteryl oligos was nearly as effective as cycloheximide at decreasing synthesis of p75, The bis-cholesteryl oligos also displayed significant efficacy at 1 mu M, whereas the other oligos required 5 mu M to be effective. The enhanced efficacy of bis-cholesteryl oligos is likely to be due to a combination of enhanced cellular uptake and resistance to both 5' and 3' exonucleases.

Molecular development of the olfactory nerve pathway

There are, at least, two major questions concerning the molecular development of the olfactory nerve pathway. First, what are the molecular cues responsible for guiding axons from the nasal cavity to the olfactory bulb? Second, what is the molecular basis of axon targeting to specific glomeruli once axons reach the olfactory bulb? Studies in the primary olfactory pathway have focused on the role of the extracellular matrix and ensheathing cells in establishing an initial substrate for growth of pioneer axons between the periphery and brain. The primary axons also express a multitude of cell adhesion molecules that regulate fasciculation of axons and hence may play a role in fascicle formation in the olfactory nerve. Although the olfactory neuroepithelium principally consists of a morphologically homogeneous class of primary olfactory neurons, there are numerous subpopulations of olfactory neurons expressing chemically distinct phenotypes. In particular, numerous subpopulations have been characterized by expression of unique carbohydrate residues and olfactory receptor proteins. Some of these molecules have recently been implicated in axon guidance and targeting to specific glomeruli.

Efficient transformation and regeneration of diverse cultivars of peanut (Arachis hypogaea L.) by particle bombardment into embryogenic callus produced from mature seeds

Peanut, one of the world's most important oilseed crops, has a narrow germplasm base and lacks sources of resistance to several major diseases. The species is considered recalcitrant to transformation, with few confirmed transgenic plants upon particle bombardment or Agrobacterium treatment. Reported transformation methods are limited by low efficiency, cultivar specificity, chimeric or infertile transformants, or availability of explants. Here we present a method to efficiently transform cultivars in both botanical types of peanut, by (1) particle bombardment into embryogenic callus derived from mature seeds, (2) escape-free (not stepwise) selection for hygromycin B resistance, (3) brief osmotic desiccation followed by sequential incubation on charcoal and cytokinin-containing media; resulting in efficient conversion of transformed somatic embryos into fertile, non-chimeric, transgenic plants. The method produces three to six independent transformants per bombardment of 10 cm(2) embryogenic callus. Potted, transgenic plant lines can be regenerated within 9 months of callus initiation, or 6 months after bombardment. Transgene copy number ranged from one to 20 with multiple integration sites. There was ca. 50% coexpression of hph and luc or uidA genes coprecipitated on separate plasmids. Reporter gene (luc) expression was confirmed in T-1 progeny from each of six tested independent transformants. Insufficient seeds were produced under containment conditions to determine segregation ratios. The practicality of the technique for efficient cotransformation with selected and unselected genes is demonstrated using major commercial peanut varieties in Australia (cv. NC-7, a virginia market type) and Indonesia (cv. Gajah, a spanish market type).

Expression of galectin-1 in the olfactory nerve pathway of rat

The olfactory neuroepithelium is characterised by the mosaic distribution of primary olfactory neurons that express different odorant receptors and cell surface glycoconjugates. Carbohydrates are believed to form a glycocode that mediates sorting out and fasciculation of primary olfactory axons through interactions with carbohydrate-binding proteins such as galectin-1. In the present study, we describe in detail the expression pattern of galectin-1 in the developing and adult rat olfactory system. We demonstrate that galectin-1 is expressed by olfactory ensheathing cells both in olfactory nerve and within the nerve fibre layer of the olfactory bulb of the embryonic and adult rat. In the adult rat, galectin-1 was preferentially expressed by olfactory ensheathing cells in the nerve fibre layer of the ventromedial and lateral surfaces of the olfactory bulb. Galectin-1 was also expressed by subsets of periglomerular cells and granule cells, particularly in the ventromedial region of the olfactory bulb. In adult rat, the galectin-1 ligand, N-acetyl-lactosamine, was expressed by primary olfactory axons that terminated in glomeruli present in the ventromedial and lateral olfactory bulb. These results suggest that expression of galectin-1 may provide a mechanism for the sorting of subpopulations of axons in the nerve fibre layer of the olfactory bulb during development as well as play a role in the postnatal maintenance of specific glomerular connections. (C) 1999 Elsevier Science B.V. All rights reserved.

Expression of EphA5 during development of the olfactory nerve pathway in rat

The olfactory neuroepithelium is a highly plastic region of the nervous system that undergoes continual turnover of primary olfactory neurons throughout life. The mechanisms responsible for persistent growth and guidance of primary olfactory axons along the olfactory nerve are unknown. In the present study, we used antibodies against the Eph-related receptor, EphA5, to localise EphA5, and recombinant EDhA5-IgG fusion protein to localise its ligands. We found that although both EphA5 and its ligands were both expressed by primary olfactory neurons within the embryonic olfactory nerve pathway, there was no graded or complementary expression pattern. In contrast, the expression patterns altered postnatally such that primary olfactory neurons expressed the ligands, whereas the second-order olfactory neurons, the mitral cells, expressed EphA5. The role of EphA5 was analysed by blocking EphA5-ligand interactions in explant cultures of olfactory neuroepithelium using anti-EphA5 antibodies and recombinant EphA5. These perturbations reduced neurite outgrowth from explant cultures and suggest that intrafascicular axon repulsion may serve to limit adhesion and optimise conditions for axon growth. (C) 2000 Wiley-Liss, Inc.

Exposure of rats to a high but not low dose of ethanol during early postnatal life increases the rate of loss of optic nerve axons and decreases the rate of myelination

Visual system abnormalities are commonly encountered in the fetal alcohol syndrome although the level of exposure at which they become manifest is uncertain. In this study we have examined the effects of either low (ETLD) or high dose (ETHD) ethanol, given between postnatal days 4-9, on the axons of the rat optic nerve. Rats were exposed to ethanol vapour in a special chamber for a period of 3 h per day during the treatment period. The blood alcohol concentration in the ETLD animals averaged similar to 171 mg/dl and in the ETHD animals similar to 430 mg/dl at the end of the treatment on any given day. Groups of 10 and 30-d-old mother-reared control (MRC), separation control (SC), ETLD and ETHD rats were anaesthetised with an intraperitoneal injection or ketamine and xylazine, and killed by intracardiac perfusion with phosphate-buffered glutaraldehyde. In the 10-d-old rat optic nerves there was a total of similar to 145000-165000 axons in MRC, SC and ETLD animals. About 4 % of these fibres were myelinated. The differences between these groups were not statistically significant. However, the 10-d-old ETHD animals had only about 75000 optic nerve axone (P < 0.05) of which about 2.8 % were myelinated. By 30 d of age there was a total of between 75000 90000 optic nerve axons, irrespective of the group examined. The proportion of axons which were myelinated at this age was still significantly lower (P < 0.001) in the ETHD animals (similar to 77 %) than in the other groups (about 98 %). It is concluded that the normal stages of development and maturation of the rat optic nerve axons, as assessed in this study, can be severely compromised by exposure to a relatively high (but not low) dose of ethanol between postnatal d 4 and 9.