Cerebrospinal fluid C3a increases with age, but does not increase further in Alzheimer's disease.

Complement activation is present in the brain in Alzheimer's disease (AD), and C1q concentrations are decreased in AD cerebrospinal fluid (CSF). To determine whether concentrations of other complement proteins are also altered in AD CSF, we measured concentrations of C3a and SC5b-9 in CSF from patients with probable AD (n = 19), normal aged controls (n = 11), and normal younger controls (n = 15). C3a concentrations were similar between AD and aged controls, but threefold higher than in younger controls (p < 0.05 vs. both groups). A similar pattern was found with SC5b-9, though the increase was only twofold and statistically significant only for AD vs. younger controls. These results suggest that an increased generation of complement proteins in localized areas of the AD brain does not result in elevated concentrations of these proteins in CSF, compared with age-matched controls. Increased C3a (and, to a lesser extent, SC5b-9) in aged controls may be due to increased complement activation, increased central nervous system production, and/or blood-brain barrier leakage of these proteins.

The novel use of vascular access ports for intravenous self-administration and blood withdrawal studies in squirrel monkeys.

An important experimental challenge in research with squirrel monkeys (Saimiri scureus) is the development of a reliable closed intravenous system for long term drug self-administration studies and the collection of multiple timed blood samples. A surgical procedure using a vascular access port (VAP) system was developed to provide easy access for venous samples or drug infusions. Daily experiments in chaired monkeys were simple and reliable for durations of up to 6 h. The quantitative performance of the VAP system was evaluated by the number of days until port failure for self-administration studies and the number of days during which blood samples could be collected beyond an initial time of 91 days. The mean best performance for VAP system functional time for self-administration studies was 437 +/- 73 and the mean worst performance was 281 +/- 79. The mean best performance for blood withdrawal functional time was 362 +/- 81 and the mean worst performance was 332 +/- 85. The qualitative performance of the VAP system is described including complications that developed during the procedure; corresponding suggestions for corrective actions are discussed. Enhancements to increase port performance are also recommended.

Increased regional brain concentrations of ceruloplasmin in neurodegenerative disorders.

Ceruloplasmin (CP), the major plasma anti-oxidant and copper transport protein, is synthesized in several tissues, including the brain. We compared regional brain concentrations of CP and copper between subjects with Alzheimer's disease (AD, n = 12), Parkinson's disease (PD, n = 14), Huntington's disease (HD, n = 11), progressive supranuclear palsy (PSP, n = 11), young adult normal controls (YC, n = 6) and elderly normal controls (EC, n = 7). Mean CP concentrations were significantly increased vs. EC (P < 0.05) in AD hippocampus, entorhinal cortex, frontal cortex, and putamen. PD hippocampus, frontal, temporal, and parietal cortices, and HD hippocampus, parietal cortex, and substantia nigra. Immunocytochemical staining for CP in AD hippocampus revealed marked staining within neurons, astrocytes, and neuritic plaques. Increased CP concentrations in brain in these disorders may indicate a localized acute phase-type response and/or a compensatory increase to oxidative stress.

The antithrombotic effects of CI-1031 (ZK-807834) and enoxaparin in a canine electrolytic injury model of arterial and venous thrombosis.

Factor Xa is a serine protease positioned at the convergence point of the intrinsic and extrinsic coagulation pathways and is therefore an attractive target in the development of novel anticoagulant drugs. The objective of this study was to evaluate the efficacy of CI-1031 (N-[2-[5-amidino-2-hydroxyphenoxy]-6-[3-(1-methyl-1H-imidazolin-2-yl)-phenoxy]-3,5-difluoropyrid), a potent and selective inhibitor of Factor Xa, in a canine electrolytic injury model of arterial and venous thrombosis. Enoxaparin (enoxaparin sodium), a low molecular weight heparin currently approved for treatment and prevention of deep vein thrombosis and unstable angina, was also tested for efficacy in this model. CI-1031 was administered intravenously to anesthetized dogs at three doses: 1.25, 2.5 and 5 microg/kg/min (n=5 for each group) as a continuous infusion for 5.5 h. The control group (n=5) received a continuous infusion of vehicle (3.69 mmol citric acid and 0.9% sodium chloride solution) at a rate of 1 ml/kg/h. Ninety minutes after administration of CI-1031 prothrombin times increased 1.2-, 1.6- and 2.0-fold over baseline values in the 1.25, 2.5 and 5 microg/kg/min groups, respectively. The time to formation of an occlusive thrombus in the femoral arteries averaged 69+/-5 min in the control group compared to 127+/-19, 192+/-33 and 219+/-15 min in the low-, mid- and high-dose CI-1031 groups. In the femoral veins, occlusion time in the controls averaged 56+/-11 min compared to 153+/-22, 137+/-30 and 214+/-26 min in the three treatment groups. Thrombus weights in the control arteries averaged 51+/-4 mg compared to 45+/-5, 28+/-10 and 15+/-3 mg in the CI-1031 treated groups. On the venous side, control thrombus weights averaged 96+/-18 mg compared to 75+/-16, 51+/-16 and 25+/-4 mg in the low-, mid- and high-dose CI-1031 groups. A plasma CI-1031 concentration of approximately 400 ng/ml was associated with a 50% reduction in thrombus weight relative to control animals. Enoxaparin was administered intravenously at a loading dose of 50, 100 or 200 IU/kg for 1 h followed by a maintenance infusion of 25, 50 or 100 IU/kg/h for 4.5 h. The most dramatic changes in coagulation parameters were observed in thrombin time with virtually no changes in prothrombin time. Enoxaparin elicited a dose-dependent increase in time to thrombotic occlusion and a dose-dependent decrease in thrombus weight similar to that observed with CI-1031. Time to occlusion in the enoxaparin-treated groups averaged 117+/-33, 188+/-32 and 217+/-22 min in the low-, mid- and high-dose groups in the femoral arteries and 84+/-22, 171+/-31 and 133+/-33 min in the femoral veins. Thrombus weights averaged 33+/-10, 12+/-5 and 10+/-4 mg in the arteries and 32+/-9, 13+/-2 and 21+/-6 mg in the veins in the low-, mid- and high-dose groups. Blood loss with CI-1031 tended to be less than enoxaparin at doses that provided comparable efficacy. These results demonstrate that CI-1031, like enoxaparin, is an effective antithrombotic agent in an established canine model of arterial and venous thrombosis. CI-1031 provided dose-dependent efficacy with minimal changes in ex vivo coagulation parameters, suggesting it may be a safe and effective antithrombotic agent for both arterial and venous indications.

Purine-induced alterations of dopamine metabolism in rat pheochromocytoma PC12 cells.

Studies with cerebrospinal fluid from subjects with Parkinson's disease suggest that purine abnormalities may be present in this disorder. The effects of purines on dopamine metabolism have not been characterized, though adenosine is known to inhibit dopaminergic neurotransmission. In this study, dopamine, its precursor 3,4-dihydroxyphenylalanine (DOPA), and its degradation products 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) were measured in rat pheochromocytoma PC12 cells following 24-h incubation with 5, 50, and 500 microM adenosine, adenine, guanosine, guanine, hypoxanthine, xanthine, and uric acid. Incubation with adenosine increased DOPA, DOPAC, and HVA, while adenine treatment decreased DOPA. Guanosine (500 microM) decreased DOPA, dopamine, and DOPAC, while lower concentrations increased DOPAC and HVA. Incubation with guanine decreased dopamine, and xanthine decreased dopamine and DOPAC. Hypoxanthine and uric acid exerted minimal effects. These results indicate that purines exert a variety of effects on dopamine metabolism. The influence of purine metabolism on the dopaminergic deficit in the Parkinsonian brain merits further investigation.

Altered guanosine and guanine concentrations in rabbit striatum following increased dopamine turnover.

The significance of guanine nucleotides and nucleosides in neurodegenerative disorders is suggested by recent reports that these molecules enhance neurite branching and astrocyte proliferation. The objective of this study was to investigate the influence of increased dopamine metabolism, produced by 5-day treatment of rabbits with reserpine (2 mg/kg) or levodopa (LD) (50 mg/kg), on striatal concentrations of guanosine, guanine, and their metabolites. Reserpine treatment decreased striatal guanosine by 41% and increased guanine by 50%, while LD decreased guanosine by 48% (all p < 0.01 vs. vehicle-treated controls). LD also increased guanine by 22% (not statistically significant). Xanthine and uric acid concentrations were unchanged. Because of the neurotrophic properties of guanosine and guanine, changes in striatal concentrations of these purines secondary to increased dopamine (DA) turnover may have relevance for survival of remaining dopaminergic neurons in Parkinson's disease (PD).

Time-dependent effects of levodopa on regional brain dopamine metabolism and lipid peroxidation.

Levodopa treatment in Parkinson's disease has been suggested to contribute to disease progression through free radical generation. We compared the time course of levodopa-induced dopamine metabolism, and the resulting oxidative stress, between rat brain regions with varying dopaminergic innervation. At 1, 4, 8, and 12 h after levodopa administration (100 mg/kg), dopamine, dihydroxyphenylacetic acid, and homovanillic acid were measured in striatum and ventral midbrain, regions containing marked dopaminergic innervation, and in prefrontal cortex and cerebellum, which possess little dopaminergic innervation. Malondialdehyde, a marker of oxidative stress, was measured in additional animals. The return of dopamine and its metabolites to control concentrations tended to be slower (by 3-8 h) in cerebellum and prefrontal cortex than in dopaminergic regions. Malondialdehyde concentrations were decreased (p < 0.05) in ventral midbrain 8 h posttreatment, but increased in cerebellum 12 h posttreatment. We concluded that levodopa increases dopamine metabolism in nondopaminergic as well as dopaminergic regions, with delayed clearance of dopamine and its metabolites in nondopaminergic regions. The slower return of dopamine to control levels in nondopaminergic regions may be relevant to some of the side effects of levodopa. No support was found for the hypothesis that levodopa treatment induces oxidative stress.

Effects of enhanced striatal dopamine turnover in vivo on glutathione oxidation.

In Parkinson's disease (PD), a compensatory increase in dopamine (DA) turnover occurs in the remaining nigrostriatal dopaminergic neurons, resulting in greater exposure of each neuron to hydrogen peroxide (H2O2) derived from oxidative deamination of DA. The formation of oxyradicals from H2O2 is regarded as a mechanism that could contribute to the progression of PD, and incubation of rat striatal synaptosomes with levodopa (LD) results in an increase in oxidized glutathione (GSSG), indicative of oxidant stress. The present study was undertaken to determine whether striatal GSSG levels increase in response to administration of LD in vivo. Acute and repeated (3-week) treatment of normal rats with LD at doses of up to 100 mg/kg did not increase striatal GSSG despite marked increase in DA turnover. These results suggest that intact striatum may possess increased defense capacity against oxidant stress generated by increased DA turnover as compared with isolated synaptosomes.

Brain extracellular levels of the putative antipsychotic CI-1007 and its effects on striatal and nucleus accumbens dopamine overflow in the awake rat.

The compound CI-1007 (R-(+)-1,2,3,6-tetrahydro-4-phenyl-1 [(3-phenyl-3-cyclohexen-1-yl)methyl]pyridine maleate) has been identified as a partial dopamine agonist and putative antipsychotic in in-vitro and in-vivo neurochemical, neurophysiological and behavioural tests. By use of microdialysis in conjunction with high-performance liquid chromatography (HPLC) with electrochemical detection, the effects of the drug on brain dopamine release, previously observed in anaesthetized animals, were shown to occur in awake animals also. Detection of peripherally administered CI-1007 in the brain, i.e. evidence of the drug's ability to penetrate the blood-brain barrier, was achieved by use of in-vivo brain microdialysis in awake, freely moving rats and capillary HPLC in combination with tandem mass spectrometry (HPLC/MS/MS). Intravenous administration of CI-1007 (40 mg kg-1) significantly inhibits dopamine release in the nucleus accumbens, a region associated with dopamine hyperactivity in schizophrenia, while having a non-significant impact on the striatal dopamine neurotransmission which is critical to regular motor function. The differential neurochemical profile of the drug indicates its potential usefulness in treating positive disease symptoms and implies that its extrapyramidal side effects are lower than those of typical antipsychotics.

Surgically induced osteoarthritis in the rat results in the development of both osteoarthritis-like joint pain and secondary hyperalgesia.

OBJECTIVE: In the present study, we sought to develop/characterize the pain profile of a rat model of surgically induced osteoarthritis (OA). METHODS: OA was surgically induced in male Lewis rats (200-225 g) by transection of the medial collateral ligament and medial meniscus of the femoro-tibial joint. In order to characterize the pain profile, animals were assessed for a change in hind paw weight distribution (HPWD), development of mechanical allodynia, and the presence of thermal and mechanical hyperalgesia. Rofecoxib and gabapentin were examined for their ability to decrease change in weight distribution and tactile allodynia. RESULTS: Transection of the medial collateral ligament and medial meniscus of male Lewis rats resulted in rapid (<3 days) changes in hind paw weight bearing and the development of tactile allodynia (secondary hyperalgesia). There was, however, no appreciable effect on thermal hyperalgesia or mechanical hyperalgesia. Treatment with a single dose of rofecoxib (10 mg/kg, PO, day 21 post surgery) or gabapentin (100mg/kg, PO, day 21 post surgery) significantly attenuated the change in HPWD, however, only gabapentin significantly decreased tactile allodynia. CONCLUSION: The rat medial meniscal tear (MMT) model mimics both nociceptive and neuropathic OA pain and is responsive to both a selective cylooxygenase-2 (COX-2) inhibitor commonly utilized for OA pain (rofecoxib) and a widely prescribed drug for neuropathic pain (gabapentin). The rat MMT model may therefore represent a predictive tool for the development of pharmacologic interventions for the treatment of the symptoms associated with OA.

Influence of tumour physico-chemical conditions on interleukin-2-stimulated lymphocyte proliferation.

The proliferative response of murine lymphocytes to interleukin-2 (IL-2) was examined under physico-chemical conditions present in solid tumours, namely low oxygen and glucose concentrations and acidic pH. Lymphocytes were cultured for four days in 30 U ml-1 IL-2 to simulate serum IL-2 concentrations attainable with high-dose systemic IL-2 therapy. Lymphocyte proliferation was significantly (P < 0.05) reduced by low oxygen concentrations (both anoxia [0% O2] and hypoxia [10%, low glucose (6 mg dl-1), or acidic pH (6.7 or 6.4). Moderate glucose concentration (32 mg dl-1), or neutral pH (7.0) did not impair proliferation. This study indicates that impairment of lymphocyte proliferation by tumour physico-chemical conditions may be a factor in the relatively poor success rate of IL-2/LAK cell immunotherapy.

Natural killer-cell activity under conditions reflective of tumor micro-environment.

Natural-killer(NK) activity was examined in the presence of low oxygen tension, low glucose concentration and acidic pH, to determine whether physical conditions present in the tumor micro-environment could play a role in down-regulating cytolytic activity of tumor-infiltrating lymphocytes with NK phenotype. Anoxia (0% O2), but not hypoxia (1% O2), significantly reduced NK activity, as did acidic pH (6.4 or 6.7). Low glucose concentration (6 mg/dl) did not impair NK activity. Combinations of either moderate (1% O2, 26 mg/dl glucose, pH 6.7) or extreme (0% O2, 6 mg/dl glucose, pH 6.4) alteration of physical conditions significantly reduced NK activity. This study indicates that the physico-chemical conditions present within solid tumors are capable of down-regulating NK activity.

Evaluation of the uncertainty of molecular mass measurement by electrospray ionization mass spectrometry.

Electrospray ionization (ES) is a novel method used in mass spectrometry (MS) for producing gas-phase ions from substances in solutions. Common practices for molecular mass estimation from ES spectra summarize the spectrum as a single peak giving no estimate of uncertainty or treat each peak as an independent molecular mass measurement. ES-MS data analysis showed that each peak in an ES spectrum does not always provide an independent measure of molecular mass. Underestimation of measurement uncertainty is a possible result. An elementary time series method, the Yule-Walker equations, was applied to molecular mass estimation from ES data.

Analysis of distribution of tumor- and preneoplasia-infiltrating lymphocytes using simultaneous Hoechst 33342 labeling and immunophenotyping.

Hoechst 33342 in vivo staining was combined with immunofluorescent staining of cell surface antigens to quantify the distribution, relative to blood supply, of lymphocytes in a preneoplastic mammary lesion, the murine C4 hyperplastic alveolar nodule (HAN), and the C4 adenocarcinoma which develops from C4 HAN. The vascular supply to lymphocytes expressing Thy 1.2, L3T4, Ly2, and ASGM1 cell surface antigens was evaluated in both tissues. The distribution of ASGM1+ cells, which include natural killer cells, differed between the two tissues, being significantly increased in the 20% brightest Hoechst-stained lymphocyte fraction in HAN but not in C4 tumor. Distribution of T lymphocytes did not differ between the two tissues. The combination of in vivo Hoechst 33342 with in vitro immunofluorescence provides a simple method to evaluate the distribution with regard to blood supply of lymphocyte subsets in solid tumors and preneoplastic lesions.

Effects of the phosphodiesterase inhibitor rolipram on streptococcal cell wall-induced arthritis in rats.

The phosphodiesterase-IV (PDE-IV) inhibitor, rolipram, is antiinflammatory in a number of animal models and inhibits the release of a variety of cytokines, including TNFalpha. Arthritis induced in rats by systemic reactivation with streptococcal cell walls (SCW) following intraarticular sensitization is a TNFalpha-dependent, delayed-type hypersensitivity (DTH) reaction. Rolipram administered during the reactivation phase dose-dependently inhibited hind paw edema through day 24, the day of peak swelling. PMN and T-cell recruitment to the arthritic joint were also attenuated in rolipram-treated rats. Histologic examination of ankle sections from rolipram-treated animals showed a marked attenuation of synovial inflammation. Mechanistic studies to determine the role of glucocorticoids in mediating rolipram action showed that the inhibitory effect of rolipram on swelling was not reversed by RU 486, a glucocorticoid antagonist. In contrast, RU 486 reversed the inhibitory effects of rolipram on TNFalpha secretion. To further evaluate the role of cAMP in the model, the beta-adrenergic receptor (betaAR) agonist isoproterenol was tested, and found to inhibit swelling but not the release of TNFalpha. These results are consistent with the view that the inhibitory effects of rolipram may be partially mediated by cAMP-dependent, but TNFalpha-independent, mechanisms. The betaAR antagonists propranolol and nadolol had no appreciable affect on the antiinflammatory effect of rolipram. However, rolipram reversed the lethal effects of the antagonists observed when either was administered alone. Apparently, beta-adrenergic mechanisms moderate the response to challenge, and rolipram treatment, presumably as a result of its effects on cAMP levels, reverses the toxic effect of the antagonists.

Effects of endothelin-receptor antagonism with PD156707 on myocardial stunning in swine.

Endothelin (ET) has been proposed to play a role in pathogenesis of myocardial ischemia/reperfusion injury. The potential role of ET in myocardial stunning has not been examined. Therefore we tested the hypothesis that selective blockade of ETA receptors with PD156707 {sodium 2-benzo[1,3]dioxol-5-yl-4-(4-methoxy-phenyl) -4-oxo-3-(3,4,5-trimethoxy-benzyl)-but-2-enoate} could improve postischemic contractile dysfunction in open-chest pigs. Myocardial stunning was achieved by a sequence of three 10-min left anterior descending (LAD) occlusions interspersed with 15 min of reperfusion. All pigs received either an intravenous saline vehicle (n =6) or PD156707 (n = 6) at a loading dose infusion of 10 mg/kg/h for 1 h before the first occlusion followed by a maintenance dose of 7 mg/kg/h for 4 h. Systolic wall thickening (percentage of baseline) was measured with sonomicrometers. There was no significant difference in systolic thickening between groups at baseline, at the end of the final stunning occlusion, or at any of the time points during reperfusion. PD156707 significantly reduced arterial blood pressure before myocardial ischemia and throughout reperfusion. There was no significant difference in size of the region at risk between groups. In conclusion, selective blockade of ETA receptors with PD156707 did not significantly alter postischemic contractile function in open-chest pigs. These results suggest that activation of ETA receptors by endogenous ET does not play a significant role in the pathogenesis of myocardial stunning.

Immunocytochemical detection of anti-hippocampal antibodies in Alzheimer's disease and normal cerebrospinal fluid.

Immunocytochemical staining was performed to investigate the presence of anti-hippocampal antibodies in cerebrospinal fluid (CSF) from patients with probable Alzheimer's disease (AD) (n = 19), aged normal controls (n = 9), and young normal controls (n = 10). Marked staining of neurons in the granule cell layer of the dentate gyrus and in pyramidal neurons in CA1-3 of the rat hippocampus was observed in 5 AD CSF samples (26%), 1 aged control sample (11%), and 1 young control sample (10%). These differences were not statistically significant. One of the immunoreactive AD CSF specimens also contained high concentrations of C5b-9, the membrane attack complex. The infrequent occurrence of anti-hippocampal antibodies in AD CSF, and the detection of similar immunoreactivity in control CSF specimens, suggest that these antibodies are unlikely to play a role in the neurodegenerative process in most individuals with AD. However, elevated C5b-9 concentration in an AD CSF specimen with marked immunoreactivity to hippocampal neurons suggests the possibility that anti-neuronal antibodies may contribute to complement activation in some AD patients.

Markers of dopamine depletion and compensatory response in striatum and cerebrospinal fluid.

Though depletion of CSF homovanillic acid (HVA) concentration has often been regarded as a direct indicator of dopamine (DA) deficiency in Parkinson's Disease (PD), CSF HVA is normal in mildly affected patients. To explore why, we measured DA and its metabolites in striatum and CSF in rabbits receiving reserpine for 5 days. Reserpine, which depletes striatal DA by disrupting vesicular storage of the neurotransmitter, results in a compensatory increase of DA turnover. In response to a 96% depletion of striatal DA, its catabolic intermediates 3,4-dihydroxyphenylacetic acid (DOPAC) and 3-methoxytyramine (3-MT) decreased 64% and 92% in striatum, although the endproduct, HVA, was unchanged. In contrast, CSF concentrations of HVA and DOPAC increased significantly, though 3-MT and levodopa (LD) were unaltered. A 5-fold rise in striatal LD concentration after reserpine-induced DA depletion provided evidence for enhanced DA synthesis. As in PD, the compensatory increase of DA synthesis after reserpine administration confounds the ability of CSF HVA to reflect DA depletion.

Weight bearing as a measure of disease progression and efficacy of anti-inflammatory compounds in a model of monosodium iodoacetate-induced osteoarthritis.

OBJECTIVE: To describe an in vivo model in the rat in which change in weight distribution is used as a measure of disease progression and efficacy of acetaminophen and two nonsteroidal anti-inflammatory drugs (NSAIDs) in a model of monosodium iodoacetate (MIA)-induced osteoarthritis (OA). METHODS: Intra-articular injections of MIA and saline were administered to male Wistar rats (175-200 g) into the right and left knee joints, respectively. Changes in hind paw weight distribution between the right (osteoarthritic) and left (contralateral control) limbs were utilized as an index of joint discomfort. Acetaminophen and two archetypal, orally administered NSAIDs, naproxen and rofecoxib, were examined for their ability to decrease MIA-induced change in weight distribution. RESULTS: A concentration-dependent increase in change in hind paw weight distribution was noted after intra-articular injection of MIA. Both naproxen and rofecoxib demonstrated the capacity to significantly (P<0.05) decrease hind paw weight distribution in a dose-dependent fashion, indicating that the change in weight distribution associated with MIA injection is susceptible to pharmacological intervention. CONCLUSION: The determination of differences in hind paw weight distribution in the rat MIA model of OA is a technically straightforward, reproducible method that is predictive of the effects of anti-inflammatory and analgesic agents. This system may be useful for the discovery of novel pharmacologic agents in human OA.