Duration of step initiation predicts freezing in Parkinson's disease.

OBJECTIVES: In some individuals with idiopathic Parkinson's disease (PD), freezing of gait episodes develops as the disease progresses. The neural mechanism underlying freezing in PD is poorly understood. Here, we report a 2-year follow-up on the novel discovery of prolonged step initiation duration as a potential marker of impending freezing. METHODS: Non-freezing PD participants in stages 2.5-4 of the Hoehn and Yahr disease severity scale were recruited from an earlier study which determined the effect of semi-virtual cues on walking. Responders were those who completed the first step faster in the presence of the virtual cues while non-responders either did not change or took longer to complete the first step. Both groups of participants were interviewed 2 years later to determine who had developed freezing of gait. RESULTS: Participants in the responder group had a 13-fold risk of developing freezing of gait within 2 years following the cueing study (OR=13.3, 95% CI=1.1-167). A cutoff score of -2.6% (i.e., a decrease in the duration of the first step with visual cues by 2.6% relative to no cues) gave a sensitivity and specificity of 100% and 89%, respectively. CONCLUSIONS: To the best of our knowledge, this is the first novel discovery of a physical predictor of freezing in PD. The time to complete the first step is a simple test to administer in the clinic or at home and may therefore be easily incorporated into a fall prevention training program for PD before the inception of freezing.

Intracerebral xenotransplantation of GFP mouse bone marrow stromal cells in intact and stroke rat brain: graft survival and immunologic response.

The present study characterized survival and immunologic response of bone marrow stromal cells (BMSCs) following transplantation into intact and stroke brains. In the first study, intrastriatal transplantation of BMSC (60,000 in 3 microl) or vehicle was performed in normal adult Sprague-Dawley male rats that subsequently received daily cyclosporin A (CsA, 10 mg/kg, IP in 3 ml) or vehicle (olive oil, similar volume) starting on day of surgery up to 3 days posttransplantation. Animals were euthanized at 3 or 30 days posttransplantation and brains were processed either for green fluorescent protein (GFP) microscopy or flow cytometry (FACS). Both GFP epifluorescence and FACS scanning revealed GFP+ BMSCs in both groups of transplanted rats with or without CsA, although significantly increased (1.6- to 3-fold more) survival of GFP+ BMSCs was observed in the immunosuppressed animals. Further histologic examination revealed widespread dispersal of BMSCs away from the graft core accompanied by many long outgrowth processes in non-CsA-transplanted animals, whereas a very dense graft core, with cells expressing only sporadic short outgrowth processes, was observed in CsA-transplanted animals. There were no detectable GFP+ BMSCs in nontransplanted rats that received CsA or vehicle. Immunologic response via FACS analysis revealed a decreased presence of cytotoxic cells, characterized by near complete absence of CD8+ cells, and lack of activation depicted by low CD69 expression in CsA-treated transplanted animals. In contrast, elevated levels of CD8+ cells and increased activation of CD69 expression were observed in transplanted animals that received vehicle alone. CD4+ helper cells were almost nondetectable in transplanted rats that received CsA, but also only minimally elevated in transplanted rats that received vehicle. Nontransplanted rats that received either CsA or vehicle displayed very minimal detectable levels of all three lymphocyte markers. In the second study, a new set of male Sprague-Dawley rats initially received bilateral stereotaxic intrastriatal transplantation of BMSCs and 3 days after were subjected to unilateral transient occlusion of middle cerebral artery. The animals were allowed to survive for 3 days after stroke without CsA immunosuppression. Epifluorescence microscopy revealed significantly higher (5-fold more) survival of transplanted GFP+ BMSCs in the stroke striatum compared with the intact striatum. The majority of the grafts remained within the original dorsal striatal transplant site, characterized by no obvious migration in intact striatum, but with long-distance migration along the ischemic penumbra in the stroke striatum. Moreover, FACS scanning analyses revealed low levels of immunologic response of grafted BMSCs in both stroke and intact striata. These results, taken together, suggest that xenotransplantation of mouse BMSCs into adult rats is feasible. Immunosuppression therapy can enhance xenograft survival and reduce graft-induced immunologic response; however, in the acute phase posttransplantation, BMSCs can survive in intact and stroke brain, and may even exhibit long-distance migration and increased outgrowth processes without immunosuppression.

The NF-kappaB inhibitor diethyldithiocarbamate (DDTC) increases brain cell death in a transient middle cerebral artery occlusion model of ischemia.

A transient ischemic middle cerebral artery occlusion model of stroke was used to examine the role of the transcription factor NF-kappaB in cell death as measured by DNA fragmentation and infarction volume. The left middle cerebral artery was occluded for either 30 min or 2 h in rats. One set of animals was pretreated with diethyldithiocarbamate (DDTC), an inhibitor of NF-kappaB, 30 min prior to reperfusion. The animals were reperfused and allowed to survive for 2 or 7 days. DNA fragmentation was assayed by in situ end labeling in the stroke core and penumbral regions. Specific cortical and subcortical regions were measured using quantitative image analysis. DNA fragmentation was seen only on the ischemic side of the brains in all cases. Overall, the DDTC-treated groups showed significantly increased DNA fragmentation within the ischemic side compared to the saline control groups. DDTC treatment also caused an increase in stroke volume based on triphenyl tetrazolium chloride staining. Electrophoretic mobility shift assays showed NF-kappaB activation peaking 15 min following reperfusion and that this activation was blocked by the DDTC treatment. This study suggests that the use of NF-kappaB inhibitors to block cell death following stroke needs to be carefully examined because global inhibitors may not promote neuronal survival.

Nuclear factor-kappa B activation during cerebral reperfusion: effect of attenuation with N-acetylcysteine treatment.

We examined activation of the transcription factor, nuclear factor-kappaB (NF-kappaB), which participates in the upregulation of endothelial cell adhesion proteins, during reperfusion after temporary middle cerebral artery occlusion (TMCAO). We hypothesized that N-acetylcysteine (NAC), an antioxidant which inhibits NF-kappaB activation, would alter events in brain reperfusion injury. We used a rat model of TMCAO. The left sides of the brains were rendered ischemic for 2 h, and then the area was allowed to reperfuse. The animals were treated with NAC (150 mg/kg) or saline placebo, sacrificed, and activated NF-kappaB was assessed in both the left and right hemispheres, all at varying intervals. Cerebral infarction volume was also measured in each of the hemispheres collected from a separate group of animals. Activated NF-kappaB, consisting of p65 and p50 Rel proteins, was significantly increased 15 min after reperfusion in the affected hemisphere. The activation at 15 min was completely abolished with NAC treatment. NAC treatment 1 h prior to the end of occlusion and at 24 h reduced the percentage infarction volume of the affected hemispheres from 35.5+/-2.8% (S.E.) to 18. 1+/-2.1% (p<0.01). NAC treatment at 1 h after the occlusion (after the NF-kappaB peak) and again at 24 h also significantly reduced the percentage infarction volume from 34.8+/-3.8% to 24.6+/-3.8% (p<0. 05). Thus, while NAC inhibited activation of NF-kappaB at 15 min after reperfusion, the drug acted to reduce cerebral infarction by additional, undefined mechanisms. These results bring into question the various roles of NF-kappaB in cerebral infarction followed by reperfusion.

Plasma membrane phospholipid and cholesterol distribution of skin fibroblasts from drug-naive patients at the onset of psychosis.

Contents of plasma membrane major phospholipids, cholesterol, and cholesteryl esters of fibroblasts from drug-naive psychotic patients were compared with those from normal controls. Total membrane lipids were extracted and individual lipids were separated on high-performance thin-layer chromatography. The contents of lipid bands were quantitated by densitometric scanning and comparing with standards. Contents of total phospholipids as well as phosphatidylserine, phosphatidylinositol and phosphatidylethanolamine were significantly lower in fibroblasts from patients than in those from normal controls (P < 0.001, < 0.005, < 0.05 respectively). Total cholesterol fraction and cholesteryl esters were also significantly lower in fibroblasts from patient (P < 0.005, < 0.001 respectively). These changes were not related to differences in age or sex. These data support the hypothesis that schizophrenia is associated with disordered membrane lipid metabolism, and that this predates the onset of psychosis.

Decreased adhesiveness and altered cellular distribution of fibronectin in fibroblasts from schizophrenic patients.

Relative to those from normal subjects, cultured skin fibroblasts from schizophrenic patients have been found to show abnormal growth characteristics and morphology. This study compared skin fibroblasts from 10 drug-free schizophrenic patients and 10 normal control subjects on cell adhesiveness to the substratum. Relative to fibroblasts from normal controls, those from patients showed significantly decreased cell adhesiveness, with no overlap in distribution between the groups. Since fibronectin, a major cell surface molecule, is known to be involved in the fibroblast adhesion to substratum, its extracellular and intracellular distribution was determined by immunocytochemical analysis. Both extracellular and intracellular levels of fibronectin were significantly lower, and the distribution was altered in fibroblasts from the patients.

Temporal changes in superoxide dismutase, glutathione peroxidase, and catalase levels in primary and peri-ischemic tissue. Monosialoganglioside (GM1) treatment effects.

Time-dependent changes in levels of the antioxidant enzymes, superoxide dismutase (SOD), glutathione peroxidase (GSHPOD), and catalase (CAT) after cortical focal ischemia in rat indicate that: (1) primary and peri-ischemic tissues differ in both rate and the magnitude of oxyradical-induced ischemic injury, and (2) ischemic tissue remains vulnerable to oxyradical damage as long as 72 h after ischemia since the antioxidant enzyme levels remain at or below basal levels. After 72 h, the increased levels of these enzymes are sufficient to protect tissue against oxyradical damage. GM1 ganglioside (10 mg/kg, im) further increased the already elevated levels of the enzymes after ischemia, thereby indicating the GM1 treatment increases the capacity of ischemic tissue to protect against oxyradical injury.

Temporal changes in edema, Na+, K+, and Ca++ in focal cortical stroke: GM1 ganglioside reduces ischemic injury.

Cortical focal ischemia in the rat was induced by middle cerebral artery occlusion (MCAo) together with permanent occlusion of the ipsilateral common carotid artery (CCAo) and a temporary (1 hr) occlusion of the contralateral CCA. By using a defined cortical tissue sampling procedure at 3, 6, 24, 72, 96, and 120 hr after the MCAo + CCAo, patterns of edema and ion (Na+, K+, and Ca++) changes in a primary and three peri-ischemic cortical areas are described. Ionic imbalances and edema formation have distinct patterns, are time dependent, and are different when comparing primary and peri-ischemic areas. Calcium increases to "neurotoxic" levels appear temporally independent of edema formation, reaching magnitudes 20 times greater than basal levels in the primary infarct area. Na+ increases correlate with increases in water, while K+ losses do not appear to be directly related to edema formation of Na+ and Ca++ increases. K+ losses are only significant in the primary infarct area. Rats treated with GM1 ganglioside (10 mg/kg, i.m.) daily showed significant reductions in edema, Na+ and Ca++ increases. These ganglioside effects were evident as early as 24 hr after the ischemic injury. Ca++ increases, which was maximal at 72 hr after the ischemic injury, was reduced by greater than 50% in GM1-treated animals. The mechanism by which GM1 is an effective neuroprotective agent may be evidenced by its effects on Ca++ influx/efflux processes in injury.

GM1 ganglioside reduces cognitive dysfunction after focal cortical ischemia.

The functional consequences of cortical focal ischemia and the effect of monosialoganglioside (GM1) treatment on learning/performance of a spatial reversal task were investigated. Cortical focal ischemia was induced by a permanent occlusion of the left common carotid artery and the ipsilateral middle cerebral artery, with a 1-h clamping of the contralateral carotid artery. Twenty-six rats were randomly assigned to three groups: sham controls, a saline-treated ischemic group, and a GM1 ganglioside-treated ischemic group (10 mg/kg/day: IM). Fifteen days after surgery rats were trained on a spatial reversal task in a two-lever operant chamber where food reward was contingent on lever pressing. Training continued from day 15 to day 21 after surgery. Cortical focal ischemia resulted in learning/performance deficits that were reduced by GM1 ganglioside treatment. The cognitive deficits were characterized by a significantly higher number of nonperseverative errors and number of responses to criterion. There was a significant difference between left and right lever performance in the saline-treated ischemic group, which was absent in shams and GM1-treated ischemic rats. On all measures GM1-treated rats were not different from sham controls.

Ganglioside reduction of ischemic injury.

Systemically administered gangliosides have been shown to be pharmacologically effective in reducing injury and facilitating recovery after CNS damage in various animal paradigms. Very recent work has indicated that ganglioside therapy following CNS ischemia in animals and in humans causes reductions in the extent of injury (acute phase) and enhanced neurological recovery (long-term effects). These studies are reviewed, and the possible mechanisms (i.e., the protection of plasma membrane integrity/function and the modulation of trophic factors) by which gangliosides protect against and reduce brain injury are discussed.

McN-A-343, a specific agonist of M1-muscarinic receptors, exerts antinicotinic and antimuscarinic effects in the rat adrenal medulla.

Pirenzepine, McN-A-343 and oxotremorine were used to determine the subtypes of muscarinic receptors involved in the secretion of catecholamines from the isolated perfused adrenal gland of the rat. In the presence of 0.1 microM pirenzepine, the concentration-secretion curve for muscarine was shifted in parallel to the right by almost one log unit. With 0.5 microM the shift was over two log units. The apparent dissociation constant for pirenzepine was about 1.12 X 10(-8) M. Perfusion with McN-A-343 (1-30 microM) did not evoke the secretion of catecholamines. A further increase to very high concentrations (100-1000 microM) caused only a modest secretion (about 50 ng/5 min with 300 microM as compared to the same amount of secretion obtained with 1 microM muscarine). Secretion evoked by nicotine was significantly reduced (30%) by 3 microM McN-A-343, and the inhibition increased (90%) with higher concentrations (100 microM). McN-A-343 also produced concentration-dependent inhibition of catecholamine secretion evoked by muscarine. A significant effect was observed at 30 microM and reached a maximum level at 300 microM. Oxotremorine, like McN-A-343 was a partial agonist on the muscarinic receptors; but unlike McN-A-343, did not block the stimulatory effects of nicotine. Although the pirenzepine data suggest that M1 receptors are responsible for the secretion of catecholamines in the rat adrenal medulla, this conclusion is not supported by the results obtained with the M1-receptor agonist, McN-A-343, which proved to be an effective blocker of muscarinic as well as nicotinic receptors.