Analysis of co-isogenic prion protein deficient mice reveals behavioral deficits, learning impairment, and enhanced hippocampal excitability.

BACKGROUND: Cellular prion protein (PrPC) is a cell surface GPI-anchored protein, usually known for its role in the pathogenesis of human and animal prionopathies. However, increasing knowledge about the participation of PrPC in prion pathogenesis contrasts with puzzling data regarding its natural physiological role. PrPC is expressed in a number of tissues, including at high levels in the nervous system, especially in neurons and glial cells, and while previous studies have established a neuroprotective role, conflicting evidence for a synaptic function has revealed both reduced and enhanced long-term potentiation, and variable observations on memory, learning, and behavior. Such evidence has been confounded by the absence of an appropriate knock-out mouse model to dissect the biological relevance of PrPC, with some functions recently shown to be misattributed to PrPC due to the presence of genetic artifacts in mouse models. Here we elucidate the role of PrPC in the hippocampal circuitry and its related functions, such as learning and memory, using a recently available strictly co-isogenic Prnp0/0 mouse model (PrnpZH3/ZH3). RESULTS: We performed behavioral and operant conditioning tests to evaluate memory and learning capabilities, with results showing decreased motility, impaired operant conditioning learning, and anxiety-related behavior in PrnpZH3/ZH3 animals. We also carried in vivo electrophysiological recordings on CA3-CA1 synapses in living behaving mice and monitored spontaneous neuronal firing and network formation in primary neuronal cultures of PrnpZH3/ZH3 vs wildtype mice. PrPC absence enhanced susceptibility to high-intensity stimulations and kainate-induced seizures. However, long-term potentiation (LTP) was not enhanced in the PrnpZH3/ZH3 hippocampus. In addition, we observed a delay in neuronal maturation and network formation in PrnpZH3/ZH3 cultures. CONCLUSION: Our results demonstrate that PrPC promotes neuronal network formation and connectivity. PrPC mediates synaptic function and protects the synapse from excitotoxic insults. Its deletion may underlie an epileptogenic-susceptible brain that fails to perform highly cognitive-demanding tasks such as associative learning and anxiety-like behaviors.

Reward and fictive prediction error signals in ventral striatum: asymmetry between factual and counterfactual processing.

Reward prediction error, the difference between the expected and obtained reward, is known to act as a reinforcement learning neural signal. In the current study, we propose a model fitting approach that combines behavioral and neural data to fit computational models of reinforcement learning. Briefly, we penalized subject-specific fitted parameters that moved away too far from the group median, except when that deviation led to an improvement in the model's fit to neural responses. By means of a probabilistic monetary learning task and fMRI, we compared our approach with standard model fitting methods. Q-learning outperformed actor-critic at both behavioral and neural level, although the inclusion of neuroimaging data into model fitting improved the fit of actor-critic models. We observed both action-value and state-value prediction error signals in the striatum, while standard model fitting approaches failed to capture state-value signals. Finally, left ventral striatum correlated with reward prediction error while right ventral striatum with fictive prediction error, suggesting a functional hemispheric asymmetry regarding prediction-error driven learning.

Metalloproteinases and tissue inhibitors of metalloproteinases in exudative pleural effusions.

The aim of this study was to assess the expression of several metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in exudative pleural effusions, and their relationship with inflammatory and fibrinolytic mediators in parapneumonic effusions. The study included 51 parapneumonic effusions (30 empyema or complicated parapneumonic, 21 noncomplicated parapneumonic), 28 tuberculous, 30 malignant and 30 transudates. Inflammatory markers (tumour necrosis factor-alpha, interleukin-8, polymorphonuclear elastase), fibrinolytic system variables (tissue plasminogen activator (PA), urokinase PA (u-PA), plasminogen activation inhibitor (PAI)-1, PAI-2), and several MMPs (MMP-1, MMP-2, MMP-8, MMP-9) and TIMPs (TIMP-1, TIMP-2) were determined by ELISA in plasma and pleural fluid. Elevated MMP-2 and TIMP-1 concentrations were observed in all the pleural fluid samples studied. The group of empyema or complicated parapneumonic effusions showed higher MMP-1, MMP-8 and MMP-9 concentrations than the remaining exudates. There was no correlation between MMP and TIMP levels in plasma and pleural fluid in this group of effusions. In parapneumonic effusions, MMP-1, MMP-8 and MMP-9 showed a positive correlation with the inflammatory markers and with u-PA and PAI-1. Moreover, there was a relationship between MMP-8 concentration in pleural fluid and pleural thickening at the end of treatment. In conclusion, elevated metalloproteinase-1, -8 and -9 expression was found in parapneumonic pleural effusions. These metalloproteinases could be implicated in the local inflammatory response existing in this group of effusions.

Matrix metalloproteinase expression is related to hemorrhagic transformation after cardioembolic stroke.

BACKGROUND AND PURPOSE: In animal models of cerebral ischemia, matrix metalloproteinase (MMP) expression was significantly increased and related to blood-brain barrier disruption, edema formation, and hemorrhagic transformation (HT). MMP inhibitors reduce HT after embolic ischemia in tissue-type plasminogen activator-treated animals. We aimed to determine the relationship between MMPs and HT after human ischemic stroke. METHODS: Serial MMP-2 and MMP-9 determinations were performed by means of ELISA in 39 cardioembolic strokes in the middle cerebral artery territory. Hemorrhagic events were classified according to clinical and CT criteria (hemorrhagic infarction [HI] and parenchymal hematoma [PH]). HT was evaluated on CT at 48 hours (early HT) and again between day 5 and 7 (late HT). RESULTS: HT was present in 41% of the patients (43.75% early HI, 25% early PH and 31.25% late HI). MMP-2 values were within normal range and were unrelated to HT. Increased expression of MMP-9 (normal range <97 ng/mL) was found among patients with and without HT (159.3+/-82 versus 143.9+/-112.6 ng/mL; P=0.64). According to HT subtypes, the highest baseline MMP-9 levels corresponded to patients with late HI (240.4+/-111.2 versus 102.5+/-76.7 ng/mL for all other patients, P=0.002). Baseline MMP-9 was the only variable associated with late HI in the multiple logistic regression model (OR 9; CI 1.46, 55.24; P=0.010). Peak of MMP-9 at the 24-hour time point (250.6 ng/mL) was found before appearance of PH. CONCLUSIONS: MMPs are involved in some subtypes of HT after human cardioembolic stroke. Baseline MMP-9 level predicts late HI and a 24-hour peak precedes early PH.

[Correlation between the expression of proinflammatory cytokines and matrix metalloproteinases in the acute phase of an ischemic stroke]. / Correlación entre la expresión de citocinas proinflamatorias y metaloproteinasas de matriz en la fase aguda del ictus isquémico.

INTRODUCTION: Proinflammatory cytokines are the main responsible for the onset of postischemic inflammatory cascade. Recently, the deleterious effect of matrix metalloproteinases (MMPs) in the acute phase of stroke has been described. Animal models suggest a link between both families. OBJECTIVE: We aimed to investigate possible relations between the MMP overproduction and proinflammatory cytokine expression after human ischemic stroke. PATIENTS AND METHODS: From all consecutive stroke patients attended during a 10 months period, we selected and prospectively studied those presenting as a cardioembolic stroke involving the MCA territory. MMP 9, MMP 2 and IL 6 were serially measured by means of ELISA at study entry and at 12, 24 and 48 hours after symptoms onset. RESULTS: A total of 39 patients were studied. A positive correlation was found between mean expression of both MMPs and IL 6 (r= 0.33, p= 0.040 for MMP 2 y r= 0.45, p= 0.004 for MMP 9). From all measured timepoints, the best obtained correlation was that of MMP 9 with IL 6 at 24 hours (r= 0.418, p= 0.010). At 24 h a peak value of IL 6 was observed. Baseline MMP 2 and MMP 9 levels showed a trend to correlate with that peak of IL 6 (r= 0.329, p= 0.061 for MMP 2 y r= 0.325, p= 0.061 for MMP 9). CONCLUSION: MMP expression correlates with the inflammatory cascade activation after acute cardioembolic stroke.

Matrix metalloproteinase expression after human cardioembolic stroke: temporal profile and relation to neurological impairment.

BACKGROUND AND PURPOSE: Uncontrolled expression of matrix metalloproteinases (MMPs) can result in tissue injury and inflammation. In animal models of cerebral ischemia, the expression of MMP-2 and MMP-9 was significantly increased. However, their role in human stroke in vivo remains unknown. Therefore, we sought to determine the temporal profile of MMP expression in patients with acute ischemic stroke and to investigate its relationship to stroke severity, location of arterial occlusion, and total infarct volume. METHODS: Serial MMP-2 and MMP-9 determinations were made in 39 patients with cardioembolic strokes that involved the middle cerebral artery territory by means of enzyme-linked immunosorbent assay. Blood samples, transcranial Doppler recordings, and National Institutes of Health Stroke Scale (NIHSS) scores were obtained at baseline and at 12, 24, and 48 hours after stroke onset. Infarct volume was measured with CT scanning at 48 hours. RESULTS: No correlation was found between MMP-2 and NIHSS score at any time point, although a close relation appeared between mean MMP-9 and final NIHSS score (r=0.486, P=0.002). MMP-9 value was the only factor associated with the final NIHSS score in the multiple logistic regression model (OR 4.54, 95% CI 1.5 to 13.75). A cut-point of MMP-9 142.18 ng/mL had a positive predictive value of 94.4% to assess a patient's NIHSS (<8 or >/=8) by the end of the study. Final MMP-2 and MMP-9 levels were significantly lower when recanalization occurred (528+/-144.3 versus 681.4+/-239.2 ng/mL, P=0.031 for MMP-2; 110.2+/-100.9 versus 244.8+/-130 ng/mL, P=0.004 for MMP-9). A positive correlation was found between mean MMP-9 and infarct volume (r=0.385, P=0.022). CONCLUSIONS: MMPs are involved in the acute phase of human ischemic stroke. MMP-9 levels are associated with neurological deficit, middle cerebral artery occlusion, and infarct volume.

Hypercoagulation and hypofibrinolysis in horses with colic and DIC.

Changes in haemostasis in horses with colic were assessed by using specific and sensitive markers of coagulation and fibrinolysis activity. Blood samples from 41 horses with severe colic and from 30 healthy control horses were tested. Diagnosis of DIC was based on the findings of at least 3 of 6 abnormalities: thrombocytopenia, prolonged clotting times (PT and APTT), increased polyclonal FDPs, decreased fibrinogen and decreased AT-III activity. Plasma thrombin-antithrombin III complexes (TAT), monoclonal fibrin degradation products fragment D (D-dimer) and monoclonal fibrinogen degradation products (FgDP) were also tested by using ELISA kits. DIC was diagnosed in 16 of 41 horses with colic. Compared to control and non-DIC colic values, TAT was significantly (P < 0.000) greater in horses with colic and DIC (Control group, mean +/- s.d. 2.6 +/- 2; non-DIC colic group, 7.5 +/- 9, and DIC colic group, 30.9 +/- 36 ng/ml). Also, D-dimer was significantly (P < 0.000) less in the DIC group when compared to control and non-DIC colic values (mean +/- s.d. 677 +/- 119, 682 +/- 220 and 399 +/- 234 ng/ml, respectively). Compared to non-DIC colic values, FgDP was significantly (P < 0.05) lower in the DIC group (363 +/- 111, 437 +/- 230 and 293 +/- 187 ng/ml respectively). Both PT and APTT showed a significant positive correlation with TAT. DIC was more common among nonsurvivors and horses with ischaemic bowel. We conclude that a hypercoagulative state was detected in horses with colic, which was stronger in horses with colic and DIC. Hypofibrinolysis was present only in horses with DIC. Therefore, marked hypercoagulation together with hypofibrinolysis are associated with DIC in horses.

Tissue factor levels and high ratio of fibrinopeptide A:D-dimer as a measure of endothelial procoagulant disorder in pre-eclampsia.

To assess coagulation activation and endothelial cell injury in normotensive and pre-eclamptic pregnant women, a comparison was made of plasma levels of tissue factor, fibronectin, fibrinopeptide A and D-dimer. Samples were taken from 50 nonpregnant women, 40 normotensive pregnant women in the third trimester and 27 women with pre-eclampsia after diagnosis and before treatment. High levels of fibrinopeptide A and D-dimer were found in pre-eclamptic women. Moreover, the ratio fibrinopeptide A:D-dimer was much greater in the pre-eclampsia group than in normotensive pregnant women. The levels of fibronectin and tissue factor were also higher in the pre-eclampsia group. The increase of tissue factor levels suggests an alteration of the extrinsic coagulation pathway in pre-eclampsia. The increase of fibrinopeptide A:D-dimer ratio shows that the activation of coagulation is associated with a relative hypofibrinolysis in pre-eclampsia.

Hemorheological, coagulative and fibrinolytic changes during autologous blood donation.

BACKGROUND: Clinical data suggest that autologous blood donation may prevent postsurgical venous thrombosis. If confirmed, this is probably due to beneficial effects in rheologic and hematologic variables which may be changed in patients as a result of repeated bleeding. STUDY DESIGN AND METHODS: To ascertain this point, we studied variations in hematological, hemorheological, coagulative and fibrinolytic parameters in 30 patients undergoing autologous blood donation. RESULTS: Whole blood viscosity (WBV), plasma viscosity and blood viscosity adjusted to 40% hematocrit, progressively and substantially decreased throughout the successive bleeding at all the shear rates considered. WBV was further reduced by presurgical hemodilution with autologous plasma which decreased the platelet and leukocyte count. The hemostasis and fibrinolysis variables, however, underwent no clinically significative changes. CONCLUSION: Repeated bleedings change most hemorheological variables. By decreasing cytocrit and viscosity, reducing aggregability and increasing blood cell deformability an optimal milieu to help prevent thrombosis is artificially created.

Evaluation of thrombin-antithrombin complexes and fibrin fragment D in carbohydrate-induced acute laminitis.

Carbohydrate-induced laminitis has been associated with decreased platelet survival, decreased blood flow to the hoof wall and with the deposition of platelets and microthrombi within venules in the dermal laminae. To evaluate further the systemic prothrombotic events occurring in the prodromal stages of laminitis, plasma samples from control and laminitis-affected ponies and horses were tested for the presence of thrombin-antithrombin (TAT) complexes and fibrin fragment D (D-dimer). No statistically significant differences between the control and laminitis-affected animals were observed for either the D-dimer or the TAT complexes. Few of the values for individual animals exceeded the reference ranges for control animals. These data indicate that the prothrombotic events observed in carbohydrate-induced laminitis may not be associated with systemic activation of the coagulation or fibrinolytic systems.

Comparison of the effects of low-molecular-weight and unfractioned heparin in horses.

Thirty healthy male horses were allotted to 3 groups and treated blindly during 4 days. Group-1 horses received unfractioned calcium heparin (100 IU/kg of body weight, SC, q 12 h). Group-2 horses received a single dose of a low-molecular-weight heparin (50 anti-Xa IU/kg, SC) every morning, and a similar volume of saline solution every evening. Group-3 horses received the vehicle (saline solution), SC, every 12 hours. Citrated and EDTA-anticoagulated blood samples were collected before starting the medication (T-0) and once daily 3 hours after each morning injection (T-3, T-27, T-51, and T-75). The PCV, hemoglobin concentration, RBC and platelet counts, and clotting times (activated partial thromboplastin time and thrombin time) were determined, and a microscopic examination to detect hemagglutination was performed. Plasma concentration of heparin was measured by use of the antifactor Xa, activity assay. Bleeding time was determined on the first and fourth days, using a double-template method. The horses given unfractioned heparin had marked agglutination of erythrocytes after the first injection that became more pronounced as treatment progressed. Also, significant decrease in PCV, hemoglobin concentration, and RBC count was observed during treatment. Platelet count was significantly decreased after the first day, and clotting times were significantly prolonged. In contrast to the horses given unfractioned heparin, those given low-molecular-weight heparin did not have any agglutination of erythrocytes during the 4 days of treatment, and there were no significant changes in PCV, hemoglobin concentration, or RBC and platelet counts. Activated partial thromboplastin time increased slightly in the horses given low-molecular weight heparin, although the values remained within reference range. Both groups of horses achieved adequate concentrations of heparin in plasma for prophylactic purposes, but those given low-molecular-weight heparin achieved those values after the first injection. Bleeding times were not significantly different between heparin-treated horses and horses given saline solution during treatment. We conclude that low-molecular-weight heparin may be used more safely and conveniently in horses, because it does not affect equine erythrocytes, platelets, or clotting and bleeding times.

Effects of two different doses of hirudin on APTT, determined with eight different reagents.

The APTT has been considered the most suitable candidate to monitor the anticoagulant activity of hirudin. However, its use is hampered by problems of standardization, which make the results heavily dependent on the responsiveness of the reagent used. Our aim was to investigate if this different responsiveness of different reagents when added in vitro is to be confirmed in an ex vivo study. Two different doses of r-hirudin (CGP 39393), 0.3 mg/kg and 1 mg/kg, were administered subcutaneously to 20 New Zealand male rabbits, and the differences in prolongation of APTT 2 and 12 h later were compared, using 8 widely used commercial reagents. All groups exhibited a significant prolongation of APTT 2 h after sc administration of hirudin, both at low and high doses. But this prolongation persisted 12 h later only when the PTTa reagent (Boehringer Mannheim) was used. In general, hirudin prolonged the APTT most with the silica-based reagents. In a further study, we compared the same APTT reagents in an in vitro study in which normal pooled plasma was mixed with increasing amount of hirudin. We failed to confirm a higher sensitivity for silica-containing reagents. Thus, we conclude that subcutaneous administration of hirudin prolongs the APTT most with the silica-based reagents, but this effect is exclusive for the ex vivo model.

Effects of recombinant hirudin on D dimer levels before and after experimental venous thrombosis.

In a randomized, blind study, the effect of saline, a low-molecular-weight heparin (Fragmin), and a recombinant hirudin (CGP 39393) on thrombin-antithrombin (TAT) complexes and D dimer (DD) levels were studied in 60 rabbits. The drugs were always injected subcutaneously 2 h before the induction of thrombosis in the jugular vein by a combination of endothelial damage and flow reduction. A sample of blood was obtained just before surgery, and a further sample was obtained 10 min after thrombus formation. No significant differences were found in TAT plasma levels between the different study agents, either before or after thrombus development. However, 2 h after drug administration DD values were significantly lower in hirudin-treated animals (292 +/- 69 vs. 372 +/- 138 ng/ml; p < 0.05). Then, after thrombus formation, DD levels significantly increased in all three groups of animals, as compared to baseline levels. But the increase was significantly higher in hirudin-treated rabbits; DD levels after thrombus development were significantly higher in this group as compared to placebo (779 +/- 188 vs. 664 +/- 152 ng/ml; p < 0.05). There are no previous reports in the literature about the effect of hirudin on DD levels. Our hypothesis is that the effect of hirudin on DD may be the result of its ability to inhibit the thrombin-induced thrombus growth. If the thrombus is not allowed to grow then it will lyse more quickly, producing more DD and the process will not be impaired by the constant deposition of fibrin.(ABSTRACT TRUNCATED AT 250 WORDS)

[Normal values for hematological and hemostatic parameters in the rabbit. Determination of new parameters for experimental models of thrombosis and hemostasis]. / Valores normales de los parámetros hematológicos y hemostáticos en el conejo. Determinación de nuevos parámetros para modelos experimentales de trombosis y hemostasia.

PURPOSES: Assessment of normal haematological values in the rabbit using a semi-automatic analyser. Evaluation of new haemostatic parameters necessary for experimental models in thrombosis and haemostasis. MATERIAL AND METHODS: 20 New Zealand male rabbits of 2-2.5 kg were anaesthetized and blood was collected from the jugular vein. The following haematological determinations were carried out using a semi-automatic analyser: red blood cells count, haemoglobin, haematocrit, indexes, white blood cells count, platelet count and mean platelet volume. The parameters used to assess the physiological haemostasis in rabbit were: bleeding time, fibrinogen, prothrombin time, activated partial thromboplastin time, thrombin time; and others not yet determined in the rabbit: antithrombin III, thrombin-antithrombin complexes and F1+2 fragments to measure the basal thrombin-formation activity, and fibrin D-dimer and fibrinogen degradation products for fibrinolysis activity. RESULTS: The haematological results obtained were similar than with other techniques, but with no stress influence. Normal values for haemostatic parameters studied are shown, including the parameters for the determination of physiological thrombin-formation and fibrinolysis activity. F1+2 fragments and fibrinogen degradation products could not be evaluated. CONCLUSIONS: The haematological results obtained were completely normal. All the haemostatic parameters studied were found useful to determine the physiological activity of coagulation and fibrinolysis in the rabbit, and specially for experimental models of hypercoagulability and hyperfibrinolisys states.

Comparative study on the antithrombotic efficacy of hirudin, heparin and a low-molecular weight heparin in preventing experimentally induced venous thrombosis.

In a random, blind study, we compared the antithrombotic potential of unfractioned heparin, a low-molecular weight heparin (LMWH) and a recombinant hirudin (r-hirudin) given subcutaneously in a jugular vein thrombosis model in rabbits. All drugs and placebo were injected subcutaneously 2 h before inducing experimental thrombosis. A good thromboprophylactic effect was obtained with either LMWH and r-hirudin as compared with placebo. By contrast, no significant differences were found between groups in bleeding time. We also found a small (non-significant) prolongation of the activated partial thromboplastin time in the r-hirudin-treated animals. As for thrombin time, r-hirudin-treated rabbits exhibited too much sensitivity, with noncoagulable end-points. In our study, r-hirudin at the dose used seemed to be as effective as LMWH is in the prophylaxis of venous thrombosis.

Comparative study on the antithrombotic efficacy of four low-molecular-weight heparins in three different models of experimental venous thrombosis.

In a randomized, blind study, both the antithrombotic efficacy (reduction of thrombus weight) and potency (anti-Xa activity) of several commercially available low-molecular-weight heparins (LMWHs) were compared with those of unfractioned heparin (UFH) and placebo. Three different thrombogenic challenges were used: venous thrombosis was induced by direct endothelial damage in 60 New Zealand rabbits (group I), intracarotid injection of bovine thrombin in an additional series of 60 rabbits (group II), or after inferior-vena-cava ligature in 60 Wistar rats (group III). The drugs were administered subcutaneously 2 h before surgery in a blind fashion. The doses recommended for clinical practice were used (adjusted by body weight), except in group II animals, in whom doses were doubled. No differences were found between UFH and most LMWHs in terms of reduction of thrombus weight in group I animals. But UFH showed a weaker antithrombotic efficacy in the other two models. Similarly, one of the LMWHs used (Clexane) proved to be not as effective as the remainder. However, only clinical studies will provide enough information to verify these differences. Additionally, our findings confirm that the antithrombotic efficacy of a given drug differs according to the stimulus used to induce venous thrombosis.

Heparin-related osteoporosis in rats. A comparative study between unfractioned heparin and a low-molecular-weight heparin.

In an animal model, the effect of a high dose of conventional heparin (2 IU/g s.c. twice a day) and a low-molecular-weight heparin (LMWH; Fragmin, 1 anti-Xa U/g once a day) was compared with that of placebo on the mineral bone mass in the femur of rats. After 33 days of treatment no differences were found in the weight of the femur. But heparin-treated rats exhibited a lower density (1,249 +/- 0.046 g/ml as compared with that in control rats (p = 0.00007) and also in LMWH-treated rats (p = 0.001). Similarly, statistically significant differences have been found in ash contents. They were higher in control rats than in heparin-treated rats (p = 0.0002), and also slightly higher than in LMWH-treated rats (p = 0.01). Our findings suggest that LMWH may have a lower osteopenic effect than that of conventional heparin.