Increasing local levels of IGF-I mRNA expression using adenoviral vectors does not alter oligodendrocyte remyelination in the CNS of aged rats.

IGF-I, a growth factor that contributes to developmental myelination, shows increased levels of expression within experimental models of remyelination. The pattern of IGF-I mRNA expression changes with the rate of remyelination, with peak levels of expression occurring earlier during rapid remyelination in young adult rats compared to the slower remyelination in old adult rats. In this study we have attempted to accelerate remyelination in old adult rats by using an IGF-expressing adenoviral vector (IGF-I-Ad) to bring forward the timing of peak level of IGF-I expression. Following injection of IGF-I-Ad into focal areas of lysolecithin-induced demyelination in the spinal white matter of old adult rats we created levels of IGF-I mRNA expression at 10 days that were considerably higher than those normally occurring at this time and more similar to those in young animals. However, despite the elevated levels of IGF-I mRNA expression there was no significant change in the extent of oligodendrocyte remyelination compared to saline controls or animals injected with an adenoviral vector expressing LacZ (NT-LacZ-Ad). There was a small increase in Schwann cell remyelination in IGF-I-Ad- and NT-LacZ-Ad-injected animals compared to saline controls. These results indicate that changing the levels of IGF-I directly within demyelinating lesions undergoing remyelination is not sufficient to alter remyelination and that the proremyelinating effects of systemically delivered IGF-I are unlikely to be due to direct effects on the oligodendrocyte lineage.

Depletion of endogenous oligodendrocyte progenitors rather than increased availability of survival factors is a likely explanation for enhanced survival of transplanted oligodendrocyte progenitors in X-irradiated compared to normal CNS.

Oligodendrocyte progenitors (OPs) survive and migrate following transplantation into adult rat central nervous system (CNS) exposed to high levels of X-irradiation but fail to do so if they are transplanted into normal adult rat CNS. In the context of developing OP transplantation as a potential therapy for repairing demyelinating diseases it is clearly of some importance to understand what changes have occurred in X-irradiated CNS that permit OP survival. This study addressed two alternative hypotheses. Firstly, X-irradiation causes an increase in the availability of OP survival factors, allowing the CNS to support a greater number of progenitors. Secondly, X-irradiation depletes the endogenous OP population thereby providing vacant niches that can be occupied by transplanted OPs. In situ hybridization was used to examine whether X-irradiation causes an increase in mRNA expression of five known OP survival factors, CNTF, IGF-I, PDGF-A, NT-3 and GGF-2. The levels of expression of these factors at 4 and 10 days following exposure of the adult rat spinal cord to X-irradiation remain the same as the expression levels in normal tissue. Using intravenous injection of horseradish peroxidase, no evidence was found of X-irradiation-induced change in blood-brain barrier permeability that might have exposed X-irradiated tissue to serum-derived survival factors. However, in support of the second hypothesis, a profound X-irradiation-induced decrease in the number of OPs was noted. These data suggest that the increased survival of transplanted OPs in X-irradiated CNS is not a result of the increases in the availability of the OP survival factors examined in this study but rather the depletion of endogenous OPs creating 'space' for transplanted OPs to integrate into the host tissue.

A model for long-term transgene expression in spinal cord regeneration studies.

In order to determine the suitability of first generation adenoviral vectors for gene delivery into spinal cord white matter, four different titres of beta-galactosidase-expressing adenovirus were injected into spinal cord white matter of adult rats. At titres > or =106 p.f.u., transgene expression was extensive but severe tissue damage was observed in the form of axon degeneration, demyelination and astrocyte loss. When < or = 105 p.f.u. were injected, only low levels of axon degeneration and demyelination were observed. beta-Galactosidase activity was detectable at 72 days and did not diminish significantly with time. The immune response in the spinal cord to 105 p.f.u. over 72 days was minimal, and indistinguishable from that to injection of buffer. A prominent immune response was observed when 107 p.f.u. was injected into the spinal cord of PVG rats, and when 105 or 107 p.f.u. was injected into AO rats. These results indicate that the immune response in PVG rats to betagal-expressing adenovirus is both strain and titre dependent. First generation adenoviral vectors, therefore, induce moderate and long-lived transgene expression with minimal tissue damage and immune response when an appropriate titre is injected into the low responder PVG rat strain, providing a suitable model for assessing the effect of gene delivery in models of spinal cord injury.

Polysialylated neural cell adhesion molecule-positive CNS precursors generate both oligodendrocytes and Schwann cells to remyelinate the CNS after transplantation.

Transplantation offers a means of identifying the differentiation and myelination potential of early neural precursors, features relevant to myelin regeneration in demyelinating diseases. In the postnatal rat brain, precursor cells expressing the polysialylated (PSA) form of the neural cell adhesion molecule NCAM have been shown to generate mostly oligodendrocytes and astrocytes in vitro (Ben-Hur et al., 1998). Immunoselected PSA-NCAM+ newborn rat CNS precursors were expanded as clusters with FGF2 and grafted into a focal demyelinating lesion in adult rat spinal cord. We show that these neural precursors can completely remyelinate such CNS lesions. While PSA-NCAM+ precursor clusters contain rare P75+ putative neural crest precursors, they do not generate Schwann cells in vitro even in the presence of glial growth factor. Yet they generate oligodendrocytes, astrocytes, and Schwann cells in vivo when confronted with demyelinated axons in a glia-free area. We confirmed the transplant origin of these Schwann cells using Y chromosome in situ hybridization and immunostaining for the peripheral myelin protein P0 of tissue from female rats that had been grafted with male cell clusters. The number and distribution of Schwann cells within remyelinated tissue, and the absence of P0 mRNAs in donor cells, indicated that Schwann cells were generated by expansion and differentiation of transplanted PSA-NCAM+ neural precursors and were not derived from contaminating Schwann cells. Thus, transplantation into demyelinated CNS tissue reveals an unexpected differentiation potential of a neural precursor, resulting in remyelination of CNS axons by PNS and CNS myelin-forming cells.

Behavioural consequences of oligodendrocyte progenitor cell transplantation into experimental demyelinating lesions in the rat spinal cord.

Glial cell transplantation has the potential to be developed into a clinical treatment for human demyelinating diseases because of its demonstrated efficacy in remyelinating experimentally demyelinated axons. As a step towards clinical application it is necessary to demonstrate that the procedure is safe and efficacious in promoting behavioural recovery. In this study we transplanted glial cell progenitors into demyelinating lesions induced by intraspinal injection of ethidium bromide in the rat. Locomotor function after transplantation was assessed using a beam-walking test that has previously been shown able to detect deficits associated with demyelination in the dorsal funiculus of the rat spinal cord. Two groups of animals with transplants were examined. In one group, spontaneous remyelination was prevented by exposure of the lesion to 40 Gy of X-irradiation; in the other, male glial cells were transplanted into nonirradiated female recipients, permitting their identification by use of a probe specific to the rat Y chromosome. Following transplantation, there was severe axon loss in a large proportion of the irradiated animals and those affected did not recover normal behavioural function. In contrast, both the small proportion of the irradiated group that sustained only mild axon loss and the nonirradiated recipients of transplants recovered normal function on our behavioural test. We conclude that glial cell transplantation is able to reverse the functional deficits associated with demyelination, provided axonal loss is minimal.

Paralysis in hedgehogs (Erinaceus europaeus) associated with demyelination.

Paraplegia affected 14 hedgehogs (Erinaceus europaeus) in a wildlife rescue hospital over a period of six months. Postmortem examination revealed demyelination in the brain and spinal cord and an inflammatory response in the meninges, choroid plexus and CNS. The peripheral nervous system was not affected. In the spleen, lungs and liver there was an accumulation of megakaryocytes and other evidence of extramedullary haemopoiesis, but there was no haematological evidence of anaemia. The pattern of disease incidence and the nature of the changes in the CNS suggest they were of viral origin, but no causal agent was isolated and the possibility of a neurotoxin cause cannot be ruled out.

Prevalence of hereditary hemochromatosis in 16031 primary care patients.

BACKGROUND: Despite evidence from screening studies in northern European populations, the prevalence of hemochromatosis in primary care populations in the United States remains speculative. OBJECTIVE: To establish the feasibility of screening for hemochromatosis and to estimate the prevalence of hemochromatosis in a large primary care population. DESIGN: Cross-sectional prevalence study. SETTING: 22 primary care practices in the Rochester, New York, area. PATIENTS: 16031 ambulatory patients without a previous diagnosis of hemochromatosis. INTERVENTION: Serum transferrin saturation screening tests were offered to all adult patients in participating primary care practices. MEASUREMENTS: Patients with a serum transferrin saturation of 45% or more on initial testing had a serum transferrin saturation test done under fasting conditions and had serum ferritin levels measured. Those who had a fasting serum transferrin saturation of 55% or more and a serum ferritin level of 200 microg/L or more with no other apparent cause were presumed to have hemochromatosis and were offered liver biopsy to confirm the diagnosis. RESULTS: 25 patients had biopsy-proven hemochromatosis; 22 patients met the clinical criteria for hemochromatosis but declined liver biopsy and were classified as having clinically proven hemochromatosis; and 23 patients had a serum transferrin saturation of 55% or more with no identifiable cause, indicating probable hemochromatosis. The prevalence of clinically proven and biopsy-proven hemochromatosis combined was 4.5 per 1000 (95% CI, 3.3 to 5.8 per 1000) in the total sample and 5.4 per 1000 (CI, 4.0 to 7.1 per 1000) in white persons. The prevalence was higher in men than in women (ratio, 1.8:1). CONCLUSIONS: Hemochromatosis is relatively common among white persons. Routine screening of white persons for hemochromatosis should be considered by primary care physicians.

Rejection of wild-type and genetically engineered major histocompatibility complex-deficient glial cell xenografts in the central nervous system results in bystander demyelination and Wallerian degeneration.

Mixed glial cell cultures prepared from neonatal wild type and mutant male mice lacking either major histocompatibility complex class I, class II or both class I and II molecules (major histocompatibility complex class I(o/o)II(o/o)), and from syngeneic male rats were transplanted into female rat spinal cord white matter. Graft survival was monitored using DNA probes specific to the Y chromosome. Survival of major histocompatibility complex class-deficient grafts was not prolonged compared to wild-type grafts and in most cases grafts could not be detected at 28 days post-transplantation, at which time syngeneic grafts were still present. However, rejection of xenografts resulted in significant bystander damage to host tissue. In recipients of wild-type and major histocompatibility complex class I(o/o) xenografts the predominant pathology was demyelination. Demyelination was also observed in recipients of major histocompatibility complex class II(o/o) and major histocompatibility complex class I(o/o)II(o/o) xenografts, however in addition there was marked collagen deposition and meningeal cell invasion. Significantly more axons had undergone Wallerian degeneration in recipients of major histocompatibility complex class II(o/o) and major histocompatibility complex class I(o/o)II(o/o) xenografts than recipients of wild-type and major histocompatibility complex class I(o/o) xenografts. These findings were interpreted as evidence of a more destructive immune response associated with rejection of grafts lacking major histocompatibility complex class II molecules. It was proposed that the difference in the severity of bystander damage may be related to the previously demonstrated ability of xenogeneic major histocompatibility complex class II molecules to activate host T cells directly, whereas xenografts lacking major histocompatibility complex class II molecules were capable of activating host T cells only by the indirect pathway.

Avian paramyxovirus type 1 infection in houbara bustards (Chlamydotis undulata macqueenii): clinical and pathologic findings.

Clinical and pathologic findings of avian paramyxovirus type 1 (PMV-1) in 19 houbara bustards (Chlamydotis undulata macqueenii) imported from Pakistan into the United Arab Emirates and one captive-bred bird are reported. Clinical signs included circling, walking backward, ataxia, opisthotonos, torticollis, recumbency, head tilt, head shaking, head tremor, tucking of head under keel, nasal discharge, conjunctivitis, and diarrhea. The length of time imported birds exhibited clinical signs varied from 4 days to 18 mo after importation. Hemagglutinating antibodies against PMV-1 were detected in the sera of all 17 birds from which blood samples were collected, and PMV-1 was isolated from pooled brain, spleen, and lung tissues from two birds with acute clinical signs. There were no distinctive gross lesions at necropsy, and histologic findings were consistent with but not pathognomonic for PMV-1. All houbara bustards managed in a captive breeding and restoration program established by the National Avian Research Center have been vaccinated against PMV-1 since October 1992, and no case of PMV-1 has been reported in this collection since that time.

Use of a rat Y chromosome probe to determine the long-term survival of glial cells transplanted into areas of CNS demyelination.

A lack of suitable markers for cells which undergo division following transplantation has hindered studies assessing the long-term survival of glial cell grafts in the CNS. A probe specific to the rat Y chromosome was used to identify male glial cells grafted into an area of ethidium bromide-induced demyelination in syngeneic adult female rat spinal cord 4 weeks, 6 months and 12 months post-transplantation. At all time points there was extensive oligodendrocyte remyelination of transplanted lesions, and graft-derived cells were present within the lesion up to 12 months post-transplantation. In order to demonstrate graft-derived oligodendrocytes in the remyelinated region at 6 and 12 months, double-labelling studies were performed using the oligodendrocyte-specific antibodies carbonic anhydrase II or phosphatidyl ethanolamine-binding protein in combination with the Y chromosome probe. It was found that the majority of oligodendrocytes in the transplanted region were graft-derived. Graft-mediated remyelination was associated with a reduction in myelin sheath thickness and increase in nodal frequency similar to that observed in spontaneous remyelination, suggesting that, like axons remyelinated spontaneously, axons remyelinated by grafted cells will be capable of secure conduction. An alteration in the immunoreactivity of oligodendrocytes from carbonic anhydrase II-negative in the unlesioned dorsal funiculus to carbonic anhydrase II-positive in the remyelinated dorsal funiculus was considered to reflect a reduction in the amount of myelin supported by each oligodendrocyte, leading to the proposal that carbonic anhydrase II immunoreactivity may provide a means of identifying areas of remyelination in normally carbonic anhydrase II-negative white matter tracts.

Oligodendrocyte precursors survive poorly and do not migrate following transplantation into the normal adult central nervous system.

Cell cultures enriched for oligodendrocyte precursors were prepared for male neonatal rat pups and transplanted into the spinal cord white matter of normal and x-irradiated syngeneic adult female rats. Transplanted cells were detected using a probe specific for the rat Y chromosome immediately after transplantation and 14 days later. In non-x-irradiated tissue, significantly fewer cells were seen at 14 days compared with time zero, and no cell migration was observed. In x-irradiated tissue, cells both survived and migrated into the surrounding grey and white matter. The observed behaviour of oligodendrocyte precursors in normal adult tissue is in contrast to their behaviour in myelin mutants and neonates, where migration and survival have been well documented (Warrington et al., 1993; Lachapelle et al., 1994), but mimics the behaviour of the O-2A progenitor-like cell line, CG4, following transplantation into similar environments (Franklin et al., 1996). The findings in this study have profound implications for the use of grafts of oligodendrocyte precursors as a therapy in human demyelinating diseases, because they indicate that grafts will need to be introduced directly into each clinically relevant area of demyelination.

Failure to achieve remyelination of demyelinated rat axons following transplantation of glial cells obtained from the adult human brain.

The ability of transplanted glial cells to myelinate axons in experimental animals offers the prospect that it may be possible to achieve remyelination in human demyelinating disease by the implantation of oligodendrocyte lineage cells. Autologous normal white matter could represent a potential source of cells whose use would avoid tissue rejection and overcome ethical and practical constraints associated with the use of fetal tissue. To determine the remyelinating potential of cells isolated from adult human CNS, a cell preparation prepared from adult human white matter which contained 56% oligodendrocytes, 3% preoligodendrocytes and 1% precursor cells was transplanted into non-repairing demyelinating lesions in immunosuppressed rats created by the injection of ethidium bromide into x-irradiated spinal cord white matter. The extent of remyelination was examined 3 and 5 weeks after transplantation. Although the transplanted oligodendrocytes survived in the area of demyelination, associated with demyelinated axons and produced myelin membranes, no myelin sheaths were produced and there was no evidence of cell migration or division. The failure of human oligodendrocytes to form myelin sheaths may reflect the poor remyelinating potential of post mitotic oligodendrocytes, and the failure of the small number of co-transplanted bipotential oligodendrocyte progenitor cells to differentiate and myelinate axons may be a consequence of lack of appropriate environmental factors within the rat lesion required for expansion and differentiation of these cells.

Fibro-cartilaginous embolism in a cat.

A nine-year-old cat was presented with a history of an acute onset of paraplegia. On the basis of the neurological examination, the lesion was localised between the fourth lumbar and third sacral segments (L4 to S3) of the spinal cord. Investigations included radiography, myelography, cerebrospinal fluid analysis, routine haematology and biochemistry, feline leukaemia virus testing and urinalysis. A definitive diagnosis was not achieved and the cat was euthanased 12 days after presentation. Post mortem examination revealed infarction of the spinal cord secondary to fibrocartilaginous embolisation. This is the first reported case of fibrocartilaginous embolism in the cat in the UK.

The effect of injection technique on the passive spread of astrocytes following transplantation into rat spinal cord white matter.

Bisbenzimide-labelled astrocytes were transplanted into the spinal white matter of the rat using three different injection techniques and the variability of the longitudinal distance over which they were found was compared 30 min later. Cells spread up to 5.02 mm and the greatest variability was seen when they were injected as a bolus (54%), compared with 26% when injected over 2 min. These results show the importance of establishing the extent of passive spread of cells and its variability when performing studies in cell migration.