The effect of deprenyl washout in patients with long-standing Parkinson's disease.

Deprenyl, an irreversible MAO-B inhibitor, is known to have a symptomatic effect in de novo patients with Parkinson's disease (PD). It has, however, not been studied thoroughly in patients with advanced PD and response fluctuations. This study evaluated the effect of washout of deprenyl in patients with long-standing PD. Eleven PD patients who were on chronic treatment with deprenyl (mean age 57 +/- 8 years, mean disease duration 8.4 +/- 2.9 years), seven with response fluctuations, were enrolled in a double-blind study of a novel MAO-B inhibitor. A deprenyl washout period of one month was required prior to initiation of treatment with the trial drug. Patients were evaluated by Unified Parkinson's Disease Rating scale (UPDRS) before and one month after the washout period. Motor function was quantified by computerized tapping speed and movement time test. Results showed that neither total UPDRS scores (22 +/- 16 vs. 18 +/- 16, respectively) nor tapping speed and movement time changed significantly (4.4 +/- 0.5 vs. 4.2 +/- 0.3 Hz and 159 +/- 45 vs. 161 +/- 40 seconds; p > 0.1, respectively). However, eight patients reported various degrees of subjective deterioration, among them were the seven fluctuating patients. Two patients first began to experience response fluctuations during the washout period. It seems that deprenyl has a symptomatic effect, especially in patients with response fluctuations, and it may postpone the appearance of fluctuations in patients with PD. Attempts to discontinue treatment with deprenyl may aggravate disease symptoms.

Diurnal changes in lung tumor clearance and their relation to NK cell cytotoxicity in the blood and spleen.

Natural killer cell cytotoxicity (NKCC) was reported to manifest a circadian rhythm, peaking during wakefulness in both human blood and rat spleen. Using F344 rats, we investigated whether such fluctuations (i) reflect changes in NK cell numbers or in cytotoxicity per cell; (ii) coincide in the blood and spleen; (iii) correspond with clearance of NK-sensitive tumor cells from the lungs and (iv) influence formation of lung metastases. Two rat groups were housed in opposite 12:12 hr lighting regimens. Two hours after the onset of light or dark, both groups were either sacrificed or intravenously inoculated with tumor cells to study the following indices: NKCC and NK cell numbers in the spleen (n = 29) and blood (n = 79), lung clearance of tumor cells (n = 142) and lung metastasis (n = 69). The tumor employed, MADB106, is an NK-sensitive mammary adenocarcinoma that metastasizes only to the lungs. The results indicated that, during the dark phase, splenic NKCC increased (37% higher lytic unit [LU](50)) mostly due to a 28.9% higher percentage of NK cells in the spleen. In contrast, blood NKCC decreased by 42.5% (LU(20)) and this decline was independent of circulating NK cell numbers, which remained constant. Lung tumor clearance increased in the dark (up to 42% lower retention 9 hr after inoculation), but no corresponding changes in the number of metastases were observed 3 weeks later. We conclude that diurnal changes in rats' NKCC are organ-specific, involve changes in both cell distribution and activity and may affect short-term in vivo indices of NK tumoricidal activity.

The pharmacokinetic profile of the "first ever" oral dose of levodopa in de novo patients with Parkinson's disease.

To understand the delay in the clinical benefit that commonly occurs after initiation of levodopa (L-Dopa) treatment, we examined the pharmacokinetic profile of L-Dopa after the first oral dose ever taken of L-Dopa/carbidopa in untreated patients with Parkinson's disease and followed these parameters after 1 month of treatment. This was performed in correlation with the clinical therapeutic effect. Plasma levels of L-Dopa were measured with use of high-performance liquid chromatography with electrochemical detection after administration of the "first ever" 125 mg L-Dopa/12.5 mg carbidopa tablet in 15 patients with de novo Parkinson's disease (mean age, 69 +/- 11 y, mean disease duration, 1.5 +/- 0.8 years). Blood samples were drawn before administration and thereafter at various intervals for a period of 4 hours. Repeated measurements after the same oral dose were performed after 1 month of continued therapy with L-Dopa/carbidopa 125/12.5 mg three times daily. Patients were clinically evaluated by unified Parkinson's disease rating scale motor scores. There was a modest clinical improvement after 1 month of continuous L-Dopa treatment (motor scores, 13.1 +/- 11.6 vs. 17.6 +/- 11.7; p < 0.01). Peak plasma L-Dopa levels and area under the curve did not differ significantly between the first-ever dose and after 1 month of continuous treatment (0.9 +/- 0.1 vs. 1.0 +/- 0.1 microg/mL and 66.0 +/- 30.9 vs. 86.2 +/- 34.9 microg/mL, respectively; p < 0.1. There was also no change in time to peak levels between measurements. Results indicate that the first-ever dose of oral L-Dopa is well absorbed and that pharmacokinetic mechanisms such as reduced absorption of L-Dopa probably do not play a major role in the initial delay in clinical response to oral L-Dopa/carbidopa in patients with Parkinson's disease. The latter phenomena may be linked to central pharmacodynamic mechanisms.

The role of glutamatergic transmission in the pathogenesis of levodopa-induced dyskinesias. Potential therapeutic approaches.

Dyskinesias are the most frequent adverse effect of chronic levodopa therapy in patients with Parkinson's disease (PD). Current pharmacological treatment for this problem is unsatisfactory. Recently, there is evidence for the role of glutamate in the basal ganglia neuronal circuitry in the generation of dyskinesias. If indeed glutamatergic overactivity beyond the dopaminergic synapses plays a role in the pathogenesis of these involuntary movements, there is hope that its suppression may be beneficial without causing loss of levodopa efficacy and parkinsonian deterioration. Indeed, NMDA receptor antagonists such as amantadine and dextrometorphan can reduce such dyskinesias. We tested the efficacy of riluzole, an inhibitor of glutamatergic transmission in the inhibition of levodopa-induced dyskinesias.

Molecular biology of dopamine-induced apoptosis : possible implications for Parkinson's disease.

The causes for the highly selective loss of dopaminergic neurons in the substantia nigra pars compacta in Parkinson's disease (PD) are still unknown. However, a major advance has been recently made with the introduction of the concept of apoptosis as the route leading this specific neuronal population to degeneration. Apoptosis, or programmed cell death (PCD), is an active, controlled program inherent in every living cell. Upon receiving certain signals, cells that are destined to die undergo a highly characteristic process of "suicide." This process consists of massive biochemical and morphological alterations, including cell shrinkage, loss of cell-to-cell contacts, blebbing of cell membranes, cytoskeletal rearrangements, and DNA condensation and fragmentation. It culminates in cell conversion to membrane-bound particles (apoptotic bodies) that are ready to be digested by neighboring macrophages (1-3).

Induction of neuronal apoptosis by Semaphorin3A-derived peptide.

Collapsin-1/Semaphorin3A (Sema3A) belongs to the secreted type III semaphorins family of axon guidance molecules with chemorepulsive activity, and is suggested to play a major role in navigating axonal networks throughout development into their correct destinations. We have previously shown that semaphorins are mediators of neuronal apoptosis and can induce neuronal death in the absence of any other apoptotic trigger. We report here that exposure of neuronal cells to a small conserved peptide derived from Sema3A initiates an apoptotic death process. Administration of this peptide to cultured chick sympathetic and mouse cerebellar granule neurons caused a marked shrinkage of their axonal network and cell death, which was characterized as apoptotic, based on nuclear staining. Attenuation of neuronal cell death was obtained by treatment with antioxidants and by vascular endothelial growth factor. Survival of neurons exposed to this peptide increased by co-treatment with caspase inhibitors. Induction of apoptosis was specific to neuronal cells, similarly to that induced by the full-length Sema3A protein. Our findings therefore suggest active participation of this conserved Sema3A-derived peptide in semaphorin-induced neuronal death process.

Levodopa--an exotoxin or a therapeutic drug?

Auto-oxidation of levodopa generates toxic metabolites, such as free radicals, semiquinones and quinones. In vitro, levodopa is a powerful toxin that is lethal to cultures of neurones. This raises the concern that levodopa may also be toxic in vivo, and that chronic treatment with levodopa could induce further damage to nigrostriatal neurones in patients with Parkinson's disease, accelerating the natural predetermined rate of disease progression. Although a few animal studies have shown that chronic levodopa may be toxic in vivo, most others report that it is not. The few available clinical studies also indicate that the course of Parkinson's disease is not accelerated by chronic systemic treatment with levodopa.

Involvement of T-complex protein-1delta in dopamine triggered apoptosis in chick embryo sympathetic neurons.

The neurotransmitter dopamine (DA) is capable of inducing apoptosis in post-mitotic sympathetic neurons via its oxidative metabolites. The differential display method was applied to cultured sympathetic neurons in an effort to detect genes whose expression is transcriptionally regulated during the early stages of DA-triggered apoptosis. One of the up-regulated genes was identified as the chick homologue to T-complex polypeptide-1delta (TCP-1delta), a member of the molecular chaperone family of proteins. Each chaperone protein is a complex of seven to nine different subunits. A full-length clone of 1.9 kilobases was isolated containing an open reading frame of 536 amino acids with a predicted molecular weight of 57,736. Comparison with the mouse TCP-1delta revealed 78 and 91% homology on the DNA and protein levels, respectively. Northern blot analysis disclosed a steady and significant increase in mRNA levels of TCP-1delta after DA administration, reaching a peak between 4 and 9 h and declining thereafter. Induction of the TCP-1delta protein levels was also observed as a function of DA treatment. Overexpression of TCP-1delta in sympathetic neurons accelerated DA-induced apoptosis; inhibition of TCP-1delta expression in these neurons using antisense technology significantly reduced DA-induced neuronal death. These findings suggest a functional role for TCP-1delta as a positive mediator of DA-induced neuronal apoptosis.

Effects of titanium prosthesis, offset and size of field of view on bone mineral density measurements using quantitative computed tomography.

To estimate the accuracy of quantitative computed tomography (QCT) as a method to measure bone mineral density (BMD) in the vicinity of a titanium prosthesis, we investigated the effects of (1) titanium prosthesis, (2) offset of the longitudinal axis of the bone to be examined from that of the gantry of the CT scanner, (3) size of the field of view (FOV) and (4) the combination of these effects on CT based measurements of mineral density of cortical and cancellous bone specimens. 14 bovine cortical bone parallelepipeds and 14 bovine cancellous bone parallelepipeds were used in this investigation. The bone specimens were scanned with and without a titanium prosthesis, when centered in the gantry of the CT scanner and offset from the axis of the gantry of the CT scanner at a distance of 14 cm. Image data were then reconstructed separately with a FOV of 10 cm and 30 cm. All BMD values taken from CT images obtained under different scanning condition were compared with the BMD values of the corresponding bone parallelepiped obtained under standard condition (centered in the gantry of the CT scanner, 10 cm FOV, without titanium prosthesis). When centered in the gantry of the CT scanner, the mean relative difference of BMD measurements caused by the presence of the titanium prosthesis was less than 1% for both cortical bone and cancellous bone. Size of the FOV had a negligible effect on BMD measurements. Offset at 14 cm, however, caused a significant difference in BMD measurements (p < 0.001). It was concluded that titanium prosthesis did not interfere with BMD measurements of cortical and cancellous bone when both the specimen and prosthesis were centered in the gantry of the CT scanner. However, the effect on BMD measurements of offset at 14 cm combined with the presence of a titanium prosthesis in bone was significant.

Weanling ventromedial hypothalamic syndrome. bone geometry and biomechanics.

The effect of growth-retarding, obesifying lesions in the ventromedial hypothalamic nucleus (VMN) on bone geometry and biomechanics was investigated in male weanling rats. The animals received bilateral, symmetrical, electrolytic lesions (VMNL rats) shortly after weanling (age 27 days); sham-operated rats served as controls (SCON). The rats were maintained for 42 postoperative days and then terminated. Body weight, nose-tail length, food intake, carcass water, and lean body mass were all significantly (p < 0.001) reduced in the VMNL group compared to SCON rats. Carcass fat, lipogenic efficiency (carcass fat % laid down/mean food intake) (both p < 0.001) and epididymal fat pad weight (p < 0.01) were significantly increased in VMNL versus SCON. Femur length, anteroposterior diameter (both p < 0.001), and mediolateral femur diameter (p < 0.01) were significantly reduced in VMNL versus SCON rats, but torque and angle of torque were comparable among the groups. VMNL rats femora also showed a significant greater maximum shear stress compared to the control animals. The reduced parameters in the VMNL rats are in good agreement with the previously demonstrated reduced plasma and pituitary growth hormone levels found in this hypothalamus preparation.

The molecular mechanism of dopamine-induced apoptosis: identification and characterization of genes that mediate dopamine toxicity.

Parkinson's disease (PD) is a progressive neurological disorder caused by rather selective degeneration of the dopaminergic (DA) neurons in the substantia nigra. Though subject to intensive research, the etiology of this nigral neuronal loss is still enigmatic and treatment is basically symptomatic. The current major hypothesis suggests that nigral neuronal death in PD is due to excessive oxidative stress generated by auto- and enzymatic oxidation of the endogenous neurotransmitter dopamine (DA), the formation of neuromelanin and presence of high concentrations of iron. We have found that DA toxicity is mediated through its oxidative metabolites. Whereas thiol-containing antioxidants provided marked protection against DA toxicity, ascorbic acid accelerated DA-induced death. Using the differential display approach, we sought to isolate and characterize genes whose expression is altered in response to DA toxicity. We found an upregulation of the collapsin response mediator protein (CRM) and TCP-1delta in sympathetic neurons, which undergo dopamine-induced apoptosis. The isolation of these genes led us to examine the expression and activity of CRM and TCP-1delta related genes. Indeed, we found a significant induction of mRNAs of the secreted collapsin-1 and the mitochondrial stress protein HSP60. Antibodies directed against collapsin-1 provided marked and prolonged protection of several neuronal cell types from dopamine-induced apoptosis. In a parallel study, using antisense technology, we found that inhibition of TCP-1delta expression significantly reduced DA-induced neuronal death. These findings suggest a functional role for collapsin-1 and TCP-1delta as positive mediators of DA-induced neuronal apoptosis.

Double-stranded hamstring tendons for anterior cruciate ligament reconstruction.

Bone-patellar tendon-bone autograft is the most commonly used tissue for ACL reconstruction; however, the harvesting of patellar tendon as a free graft can lead to significant morbidity. Alternate grafts may lower morbidity, yet the most commonly used alternate grafts including the double-stranded semi-tendinosus or gracilis have not been studied biomechanically. This study investigated the morphometric and biomechanical properties of double-stranded semi-tendinosus and gracilis tendons separately along with the patellar and quadriceps tendons obtained bilaterally from six fresh, 77-year-old cadaveric specimens. The quadriceps tendon was the thickest and therefore had the largest cross-sectional area, whereas double-stranded semitendinosus had the highest mean peak load to failure (1029+/-158.4 N), 11.5% and 10.3% stronger than patellar tendon and quadriceps tendons, respectively. Midsubstance rupture occurred in the hamstring tendons, whereas the patellar and quadriceps tendons failed at the bone-tendon junctions. Semitendinosus tendons with higher cross-sectional area had higher peak loads to failure. This linear relationship between cross-sectional area and the peak load to rupture also was observed in the other tendon groups (except gracilis). These results indicate that despite a lower cross-sectional area of the double-stranded semitendinosus, this tendon demonstrated a comparable mean peak load to rupture and stress compared with patellar and quadriceps tendons. It also was demonstrated that combined double-stranded semitendinosus and gracilis tendons produce a stronger graft with initial strength twice that of the patellar tendon, but requires further testing.

Semaphorins as mediators of neuronal apoptosis.

Shrinkage and collapse of the neuritic network are often observed during the process of neuronal apoptosis. However, the molecular and biochemical basis for the axonal damage associated with neuronal cell death is still unclear. We present evidence for the involvement of axon guidance molecules with repulsive cues in neuronal cell death. Using the differential display approach, an up-regulation of collapsin response mediator protein was detected in sympathetic neurons undergoing dopamine-induced apoptosis. A synchronized induction of mRNA of the secreted collapsin-1 and the intracellular collapsin response mediator protein that preceded commitment of neurons to apoptosis was detected. Antibodies directed against a conserved collapsin-derived peptide provided marked and prolonged protection of several neuronal cell types from dopamine-induced apoptosis. Moreover, neuronal apoptosis was inhibited by antibodies against neuropilin-1, a putative component of the semaphorin III/collapsin-1 receptor. Induction of neuronal apoptosis was also caused by exposure of neurons to semaphorin III-alkaline phosphatase secreted from 293EBNA cells. Anti-collapsin-1 antibodies were effective in blocking the semaphorin III-induced death process. We therefore suggest that, before their death, apoptosis-destined neurons may produce and secrete destructive axon guidance molecules that can affect their neighboring cells and thus transfer a "death signal" across specific and susceptible neuronal populations.

Current management of motor fluctuations in patients with advanced Parkinson's disease treated chronically with levodopa.

Motor fluctuations after long-term administration of levodopa may be due to central pharmacodynamic mechanisms such as reduced striatal synthesis and storage of dopamine from exogenous levodopa and subsensitization of postsynaptic dopaminergic receptors. Peripheral pharmacokinetic mechanisms may be equally important, particularly in motor fluctuations of the "delayed on" (increased time latencies from dose intake to start-up of clinical benefit) and "no-on" (complete failure of a levodopa dose to exert an "on" response) types. Levodopa itself has a very poor solubility. In addition, there is delayed gastric emptying in many advanced patients. Therefore, an oral dose of levodopa may remain in the stomach for long periods of time before it passes into the duodenum where there is immediate absorption. Consequently, in order to overcome response fluctuations caused by impaired pharmacokinetic mechanisms and to improve its absorption, we recommend that levodopa be taken in multiple small doses, on an empty stomach, preferably crushed and mixed with a lot of liquid. Protein intake should be minimized. Prokinetic drugs such as prepulsid (Cisaprid) could be used to facilitate gastric motility and levodopa transit time. Administration of crushed levodopa through nasoduodenal or gastrojejunostomy tubes may be helpful in certain circumstances. Bypassing the stomach with subcutaneous injections of apomorphine may provide dramatic rescue from difficult "off" situations. Oral and s.c. administration of novel, extremely soluble prodrugs of levodopa, e.g., levodopa ethylester, may offer a new approach to overcome difficulties in levodopa absorption. Addition of dopamine agonists, MAO-B inhibitors, COMT inhibitors and controlled release levodopa preparations may be helpful in prolonging the duration of efficacy of each single levodopa dose. Levodopa, administered orally, usually combined with peripheral dopa decarboxylase inhibitors, continues to be the most widely-used and most effective pharmacological treatment for Parkinson's disease (Melamed, 1987). Undoubtedly, the outstanding therapeutic success of levodopa represents a dramatic and revolutionary breakthrough in medicine, in general, and in neurology, in particular. Although, since the introduction of levodopa, there have been many additional pharmacological and even surgical anti-parkinsonian strategies, it still stands out as a mandatory axis of treatment in the majority of patients (Steigler and Quinn, 1992). Indeed, levodopa therapy improves, sometimes markedly, the motor signs and symptoms of the illness, the functional capacity and quality of life and perhaps also life expectancy of the afflicted patients. It is therefore unfortunate that after an initial problem-free period of successful, smooth and stable clinical benefit from levodopa that lasts about two to five years, the responsiveness of many patients worsens with the emergence of a variety of complications (Marsden et al., 1982; Hardie et al., 1984). These adverse reactions include dyskinesias and dystonias, psychotic problems and, particularly, the troublesome motor fluctuations (Marsden and Parkes, 1977; Marsden, 1994). The latter phenomenon may be particularly complex, limiting and disabling. It is believed that most patients on long-term levodopa therapy will, sooner or later, develop response fluctuations of varying types and severity (Riley and Lang, 1993). Because of the serious impact of these phenomena on the quality of life and function of the patients, many efforts are now being undertaken to identify the responsible mechanisms and to devise preventive and therapeutic measures.

Reduced femoral geometry but normal biomechanics in the dorsomedial hypothalamic nucleus-lesioned rat.

Bone geometry, structure, and biomechanical properties were investigated in a model of growth retardation, the dorsomedial hypothalamic nucleus-lesioned (DMNL) weanling rat. Male weanling Sprague-Dawley rats received bilateral electrolytic lesions in the dorsomedial hypothalamic nucleus (DMN) at age 27 days. Sham-operated rats served as controls. All rats were maintained postoperatively for 40 days. Upon sacrifice, DMNL rats weighed less (p < 0.01), were shorter (p < 0.01), and ate less (p < 0.01) when compared to controls, but their body composition was normal. The femora in DMNL were shorter (p < 0.01), had a smaller outer anteroposterior (AP) diameter (p < 0.04), polar moment of the area (p < 0.02), and maximal (p < 0.02) and minimal (p < 0.03) principal moment of the area when compared with sham-operated rats. Notably, mean torque at failure, torsional energy, stiffness, and maximal stress did not demonstrate statistically significant differences between the two groups. These data clearly show that despite the reduced size and bone growth, DMNL rats responded normally to the mechanical challenges applied to test bone biomechanical properties. The data, therefore, add to previous evidence and strengthen the hypothesis that DMNL rats are governed by an "organismic" set point.

The involvement of p53 in dopamine-induced apoptosis of cerebellar granule neurons and leukemic cells overexpressing p53.

1. The pathogenesis of the selective degeneration of the dopaminergic neurons in Parkinson's disease is still enigmatic. Recently we have shown that dopamine can induce apoptosis in postmitotic neuronal cells, as well as in other cellular systems, thus suggesting a role for this endogenous neurotransmitter and associated oxidative stress in the neuronal death process. 2. Dopamine has been shown to be capable of inducing DNA damage through its oxidative metabolites. p53 is a transcription factor that has a major role in determining cell fate in response to DNA damage. We therefore examined the possible correlation between dopamine-triggered apoptosis, DNA damage and levels of total phosphorylated p53 protein in cultured mouse cerebellar granule neurons. 3. Marked DNA damage and apoptotic nuclear condensation and fragmentation were detected within several hours of exposure to dopamine. An associated marked threefold increase in p53 phosphorylation was observed within this time window. Using a temperature-sensitive p53 activation system in leukemia LTR6 cells, were found that p53 inactivation dramatically attenuated dopamine toxicity. 4. We therefore conclude that DNA damage and p53 activation may have a role in mediating dopamine-induced apoptosis. Modulation of the p53 system may therefore have a protective role against the toxicity of this endogenous neurotransmitter and associated oxidative stress.

Early-occurrence of manual motor blocks in Parkinson's disease: a quantitative assessment.

OBJECTIVES: Quantitative analysis of internally-cued, repetitive motor performance in patients with Parkinson's disease (PD). MATERIAL AND METHODS: Computerized quantitative measurements of the frequency, duration and temporal profile of manual motor blocks (MMBs) during performance of a manual tapping task, in 39 patients with PD, as compared to 17 age-matched healthy controls. RESULTS: Performance of PD patients was markedly abnormal both quantitatively and qualitatively, as reflected by an increase (7.0% vs. 4.6%) in MMBs, and by their occurrence from onset of movement. The phenomenon was already observed in the early stages of the disease, and was also correlated with the occurrence of freezing of gait. A standard levodopa-carbidopa (125-12.5 mg) dose only partially affected this phenomenon. CONCLUSIONS: Augmented motor blocks in internally-cued performance should be recognized as a frequent, distinct and generalized feature of PD. Whereas the gait disorder is regarded as characteristic of the advanced stage of PD, our findings suggest that the basic defect in internal rhythm formation can be detected by sensitive measurement tools from the early stages of the disease. In addition, the methodology developed in this study to quantitatively measure manual motor blocks may be a useful tool for future development of therapeutic regimens for this debilitating aspect of motor dysfunction in PD.