Restorative Neurology and Neuroscience - Volume 21, issue 1-2

Authors: Phillips, André W. | Zhang, Peisu | Truckenmiller, Mary Ellen | Keir, Stuart D. | Bouvier, Margaret | Tornatore, Carlo | Freed, William J.

Article Type: Research Article

Abstract: Purpose: Adeno-associated virus (AAV) can infect a wide variety of mammalian cell types and is capable of infecting both dividing and non-dividing cell populations. Here we report the construction of a recombinant AAV vector which expresses the SV40 large T protein (AAV-T) and the use of this vector to immortalize primary cells from embryonic rat mesencephalon. Methods: The AAV-T vector was constructed by introducing the BamH1 fragment of the pCMV/SVE/Neo plasmid containing T antigen and SV40 regulatory …elements into the JM48 plasmid containing the inverted terminal repeats of AAV. Neuronal cultures from E-12 rat mesencephalon were grown in defined media supplemented with basic fibroblast growth factor. These cells were infected with the AAV-T vector. Results: A cell line (designated RMAT) and six subclones were established from these cultures through multiple passages. This cell line was immunoreactive for SV40 large T antigen and the cytoskeletal proteins nestin and vimentin. Morphological differentiation and expression of neurofilament 160 kDa were induced by exposure to dibutyrl cyclic AMP. Immunoassays performed to measure endogenous production of growth factors showed that RMAT cells produced high levels of platelet-derived growth factor (PDGF). Conclusions: AAV may be a useful vector for the transduction of oncogenes to produce cell lines. Show more

Keywords: adeno-associated virus, AAV, SV40 large T, neurotrophic factors, PDGF

Citation: Restorative Neurology and Neuroscience, vol. 21, no. 1-2, pp. 1-10, 2003

Authors: Schuetz, Erik | Rose, Karin | Thanos, Solon

Article Type: Research Article

Abstract: Purpose: To compare the effect of cutting the optic nerve versus replacing the cut optic nerve with a peripheral nerve (PN) graft on retinal glial markers, and to determine whether the PN graft can stabilize regenerating retinal ganglion cells (RGCs), thus preventing their death following re-axotomy. Methods: Retinas harvested after ganglion cell regeneration into a sciatic nerve graft were compared to untreated control retinas and retinas obtained following optic nerve axotomy. Glial-specific proteins such as …glial fibrillary acidic protein (GFAP), Bcl-2 and complement-3 receptor (Ox-42) were examined using immunohistochemistry. Ganglion cells that survived the second axotomy were quantified on retinal flat mounts by retrograde labeling from the graft. Results: GFAP expression in astrocytes and Muller cells was elevated in axotomized retinas when compared to controls, and an additional up-regulation in Muller cells was found in retinas following ganglion cell regeneration. Increased GFAP expression in retinas containing regenerated neurons was accompanied by increased Bcl-2 expression with latter being confined to Muller cells. Moreover, re-axotomy of the regenerated axons within the graft did not result in significant retrograde degeneration of RGCs within 28 days. Conclusions: The data suggest that the graft stabilizes the regenerating RGCs to an extent reminiscent of peripheral neurons, a process that may involve the interaction between neuronal and glial elements. Show more

Keywords: nerve injury, axonal regeneration, nerve grafting, rat retina

Citation: Restorative Neurology and Neuroscience, vol. 21, no. 1-2, pp. 11-18, 2003

Authors: Julkunen, Laura | Tenovuo, Olli | Jääskeläinen, Satu | Hämäläinen, Heikki

Article Type: Research Article

Abstract: Purpose. Our aim was to study whether homonymous visual field defects could be restored in chronic stroke patients (N = 5) using computer-assisted training, and whether the possible beneficial effect could be maintained. Visual evoked potential (VEP) recordings were applied to evaluate whether they could demonstrate the possible training effects at the cortical level. Methods. We applied a specially designed computer program in the training. Subjective assessment, static and kinetic perimetry, and pattern …reversal hemifield VEP recordings were used to detect the possible changes. Results. Two patients showed improvements immediately after the training in static perimetry. Further improvements were detected at a later follow-up in three patients. With kinetic perimetry, improvements were detected in three patients. The visual field defect was detected with VEP recordings in four patients before the training, and improvements could be verified with the method in three patients, two of which clearly improved in the static perimetry as well. Conclusions. In three patients, the visual defect diminished with the computer-assisted method. It seems evident that visual field defects resulting from stroke can be partially restored even in the chronic phase. Show more

Keywords: visual field defect, chronic stroke, rehabilitation, computer-assisted training, perimetry, visual evoked potential, plasticity

Citation: Restorative Neurology and Neuroscience, vol. 21, no. 1-2, pp. 19-28, 2003

Authors: Matarredona, Esperanza R. | Meyer, Morten | Seiler, Rolf W. | Widmer, Hans R.

Article Type: Research Article

Abstract: Purpose: To analyze the effects of CGP 3466, a compound structurally related to deprenyl, on survival and function of fetal ventral mesencephalic dopaminergic neurons. Methods: Free-floating roller tube (FFRT) cultures of rat (E14) ventral mesencephalon were treated with CGP 3466 [10-8 M] for 7 days. Tyrosine hydroxylase (TH) and glial fibrillary acidic protein (GFAP) immunocytochemistry was performed to allow analysis of dopaminergic neurons and astroglial cells, respectively. Lactate dehydrogenase activity in the culture medium …served as a measure of cell death. Control and CGP 3466 treated cultures were grafted into 6-hydroxydopamine-lesioned rats, and graft survival and function evaluated 9 weeks posttransplantation. Results: FFRT cultures treated with CGP 3466 contained significantly more (two fold) surviving TH-immunoreactive (-ir) neurons and decreased lactate dehydrogenase activity in the culture medium compared to controls. The actions of CGP 3466 seem not to be mediated by astroglial cells, since GFAP-ir cell numbers and GFAP protein levels did not differ between the groups. CGP 3466 pretreatment of donor cultures did not improve TH-ir cell survival in the grafts nor did it alter functional recovery as compared to controls. Conclusions: CGP 3466 is an effective survival factor for cultured midbrain dopaminergic neurons. However, CGP 3466 pretreatment does not ameliorate survival and function of the dopaminergic neurons after grafting into a rat model of Parkinson's disease. Show more

Keywords: tissue culture, rat, deprenyl, Glial cell line-derived neurotrophic factor, tyrosine hydroxylase, neural transplantation, Parkinson's disease

Citation: Restorative Neurology and Neuroscience, vol. 21, no. 1-2, pp. 29-37, 2003

Authors: You, Si-Wei | Chen, Bing-Yao | Liu, Hui-Ling | Lang, Bing | Xia, Jie-Lai | Jiao, Xi-Ying | Ju, Gong

Article Type: Research Article

Abstract: Purpose: A major issue in analysis of experimental results after spinal cord injury is spontaneous functional recovery induced by remaining nerve fibers. The authors investigated the relationship between the degree of locomotor recovery and the percentage and location of the fibers that spared spinal cord transection. Methods: The spinal cords of 12 adult rats were transected at T9 with a razor blade, which often resulted in sparing of nerve fibers in the ventral spinal cord. …The incompletely-transected animals were used to study the degree of spontaneous recovery of hindlimb locomotion, evaluated with the BBB rating scale, in correlation to the extent and location of the remaining fibers. Results: Incomplete transection was found in the ventral spinal cord in 42% of the animals. The degree of locomotor recovery was highly correlated with the percentage of the remaining fibers in the ventral and ventrolateral funiculi. In one of the rats, 4.82% of remaining fibers in unilateral ventrolateral funiculus were able to sustain a certain recovery of locomotion. Conclusions: Less than 5% of remaining ventrolateral white matter is sufficient for an unequivocal motor recovery after incomplete spinal cord injury. Therefore, for studies with spinal cord transection, the completeness of sectioning should be carefully checked before any conclusion can be reached. The fact that the degree of locomotor recovery is correlated with the percentage of remaining fibers in the ventrolateral spinal cord, exclusive of most of the descending motor tracts, may imply an essential role of propriospinal connections in the initiation of spontaneous locomotor recovery. Show more

Keywords: spinal cord transection, remaining spinal fibers, white matter, ventrolateral funiculus, locomotion, rat

Citation: Restorative Neurology and Neuroscience, vol. 21, no. 1-2, pp. 39-45, 2003

Authors: Khalyfa, Abdelnaby | Carlson, Bruce M. | Dedkov, Eduard I. | Wang, Eugenia

Article Type: Research Article

Abstract: Purpose: This study was designed to determine whether the quantitative relationship between the levels of the eEF1A-1(developmental) and eEF1A-2/S1 (adult) isoforms of peptide elongation factor remains stable after denervation of skeletal muscle or whether in response to denervation the relative amount of the developmental form would increase. In normal postnatal rat muscle, eEF1A-2/S1 is the dominant form represented, and levels of eEF1A-1 are extremely low. Methods: One hind limb in young adult rats was …permanently denervated. Denervated and corresponding contralateral control muscles were removed for biochemical and morphological analysis from 2 days to 25 months after denervation. Results: By one month after denervation, relative levels of eEF1A-1 rose dramatically in relation to those of eEF1A-2/S1, and they remained high throughout the remainder of the 25-month denervation period. Ultrastructural analysis showed a complex mix of muscle fiber atrophy, dying muscle nuclei and muscle fibers, and newly forming muscle fibers in the same tissue. Conclusions: As during muscle regeneration, levels of the developmental eEF1A-1 isoform of peptide elongation factor greatly increased relative to those of the adult eEF1A-2/S1 adult isoform following denervation in rat muscles. However, in contrast to regeneration, the elevated level of eEF1A-1 did not return to the basal minimal level. Since switching from eEF1A-1 to eEF1A-2/S1 is an indicator that terminal differentiated is completed, the failure of eEF1A-1 to return to basal level may be indicative of the persistence of an unstable tissue environment that includes muscle fiber atrophy, degeneration and neomyogenesis. The specific cellular basis for the increase in eEF1A-1 could not be determined from this study. Show more

Keywords: skeletal muscle, denervation, peptide elongation factors, ultrastructure, rat

Citation: Restorative Neurology and Neuroscience, vol. 21, no. 1-2, pp. 47-53, 2003

Authors: Hentschel, S. | Lewerenz, A. | Nieber, K.

Article Type: Research Article

Abstract: Purpose: The aim of our study was to characterize the influence of A_3 receptors on synaptic potentials (PSPs) in pyramidal cells from the rat cingulate cortex during hypoxia. METHODS: Intracellular recordings ($n=49$) were taken from slice preparations. PSPs were evoked by electrical stimulation. Results: Hypoxia (95%NA_2 -5%COA_2 , 5 min) reduced the amplitude of the PSPs significantly. The effect was more pronounced in the presence of …adenosine re-uptake inhibitor S-(p-nitrobenzyl)-6-thioguanosine (NBTG) and deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA); the effect was completely reversed by bovine adenosine deaminase. Hypoxic inhibition induced after A_1 receptor blockade with 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) in the presence of NBTG was completely reversed by the A_3 antagonist 9-chloro-2-(2-furanyl)-5-[(phenylacetyl)amino]-1,2,4-triazolo[1,5-c]quinazoline (MRS 1220), indicating the involvement of A_3 receptors in hypoxic PSP inhibition. This was confirmed by A_3 agonist NA^6 -(3-iodobenzyl)-adenosine-5'-N-methylcarboxamide (IB-MECA) inhibiting PSPs. The effect of IB-MECA was blocked by the rat A_3 receptor-selective antagonist 3-propyl-6-ethyl-5-[(ethylthio)carbonyl]-2-phenyl-4-propyl-3-pyridinecarboxylate (MRS 1523) and was not observed in the presence of G-protein inhibitor guanosine-5'-O-2-thiodiphosphate (GDP-β-S). Conclusion: We conclude that a high level of endogenous adenosine, which occurs during hypoxia, activates A_3 receptors. Their activation contributes to PSP inhibition by adenosine during hypoxia. Show more

Keywords: adenosine, A_3 receptor, hypoxia, synaptic potentials, cortex, pyramidal cells

Citation: Restorative Neurology and Neuroscience, vol. 21, no. 1-2, pp. 55-63, 2003

Article Type: Other

Citation: Restorative Neurology and Neuroscience, vol. 21, no. 1-2, pp. 65-73, 2003