Apoptosis in experimental cerebral malaria: spatial profile of cleaved caspase-3 and ultrastructural alterations in different disease stages

Cerebral malaria (CM) is associated with high mortality and morbidity as a certain percentage of survivors suffers from persistent neurological sequelae. The mechanisms leading to death and functional impairments are yet not fully understood. This study investigated biochemical and morphological markers of apoptosis in the brains of mice infected with Plasmodium berghei ANKA. Cleaved caspase-3 was detected in the brains of animals with clinical signs of CM and immunoreactivity directly correlated with the clinical severity of the disease. Caudal parts of the brain showed more intense immunoreactivity for cleaved caspase-3. Double-labelling experiments revealed processing of caspase-3 primarily in neurons and oligodendrocytes. These cells also exhibited apoptotic-like morphological profiles in ultrastructural analysis. Further, cleavage of caspase-3 was found in endothelial cells. In contrast to neurons and oligodendrocytes, apoptosis of endothelial cells already occurred in early stages of the disease. Our results are the first to demonstrate processing of caspase-3 in different central nervous system cells of animals with CM. Apoptosis of endothelial cells may represent a critical issue for the development of the disease in the mouse model. Neurological signs and symptoms might be attributable, at least in part, to apoptotic degeneration of neurons and glia in advanced stages of murine CM.

Behavioural and histopathological alterations in mice with cerebral malaria.

Different features of sensorimotor function and behaviour were studied in murine cerebral malaria (CM) and malaria without cerebral involvement (non-CM) applying the primary screen of the SHIRPA protocol. Histopathological analysis of distinct brain regions was performed and the relative size of haemorrhages and plugging of blood cells to brain vasculature was analysed. Animals suffering from CM develop a wide range of behavioural and functional alterations in the progressive course of the disease with a statistically significant impairment in all functional categories assessed 36 h prior to death when compared with control animals. Early functional indicators of cerebral phenotype are impairments in reflex and sensory system and in neuropsychiatric state. Deterioration in function is paralleled by the degree of histopathological changes with a statistically significant correlation between the SHIRPA score of CM animals and the mean size of brain haemorrhage. Furthermore, image analysis yielded that the relative area of the brain lesions was significantly larger in the forebrain and brainstem compared with the other regions of interest. Our results indicate that assessment of sensory and motor tasks by the SHIRPA primary screen is appropriate for the early in vivo discrimination of cerebral involvement in experimental murine malaria. Our findings also suggest a correlation between the degree of functional impairment and the size of the brain lesions as indicated by parenchymal haemorrhage. Applying the SHIRPA protocol in the functional characterization of animals suffering from CM might prove useful in the preclinical assessment of new antimalarial and potential neuroprotective therapies.

Initiation of high-frequency oscillatory ventilation and its effects upon cerebral circulation in pigs: an experimental study

BACKGROUND: Current practice at high-frequency oscillatory ventilation (HFOV) initiation is a stepwise increase of the constant applied airway pressure to achieve lung recruitment. We hypothesized that HFOV would lead to more adverse cerebral haemodynamics than does pressure controlled ventilation (PCV) in the presence of experimental intracranial hypertension (IH) and acute lung injury (ALI) in pigs with similar mean airway pressure settings. METHODS: In 12 anesthetized pigs (24-27 kg) with IH and ALI, mean airway pressure (P(mean)) was increased (to 20, 25, 30 cm H(2)O every 30 min), either with HFOV or with PCV. The order of the two ventilatory modes (cross-over) was randomized. Mean arterial pressure (MAP), intracranial pressure (ICP), cerebral perfusion pressure (CPP), cerebral blood flow (CBF) (fluorescent microspheres), cerebral metabolism, transpulmonary pressures (P(T)), and blood gases were determined at each P(mean) setting. Our end-points of interest related to the cerebral circulation were ICP, CPP and CBF. RESULTS: CBF and cerebral metabolism were unaffected but there were no differences between the values for HFOV and PCV. ICP increased slightly (HFOV median +1 mm Hg, P<0.05; PCV median +2 mm Hg, P<0.05). At P(mean) setting of 30 cm H(2)O, CPP decreased during HFOV (median -13 mm Hg, P<0.05) and PCV (median -17 mm Hg, P<0.05) paralleled by a decrease of MAP (HFOV median -11 mm Hg, P<0.05; PCV median -13 mm Hg, P<0.05). P(T) increased (HFOV median +8 cm H(2)O, P<0.05; PCV median +8 cm H(2)O, P<0.05). Oxygenation improved and normocapnia maintained by HFOV and PCV. There were no differences between both ventilatory modes. CONCLUSIONS: In animals with elevated ICP and ALI, both ventilatory modes had effects upon cerebral haemodynamics. The effects upon cerebral haemodynamics were dependent of the P(T) level without differences between both ventilatory modes at similar P(mean) settings. HFOV seems to be a possible alternative ventilatory strategy when MAP deterioration can be avoided.

Effect of hydration status on cerebral blood flow and cerebrospinal fluid lactic acidosis in rabbits with experimental meningitis

The effects of hydration status on cerebral blood flow (CBF) and development of cerebrospinal fluid (CSF) lactic acidosis were evaluated in rabbits with experimental pneumococcal meningitis. As loss of cerebrovascular autoregulation has been previously demonstrated in this model, we reasoned that compromise of intravascular volume might severely affect cerebral perfusion. Furthermore, as acute exacerbation of the inflammatory response in the subarachnoid space has been observed after antibiotic therapy, animals were studied not only while meningitis evolved, but also 4-6 h after treatment with antibiotics to determine whether there would also be an effect on CBF. To produce different levels of hydration, animals were given either 50 ml/kg per 24 h of normal saline ("low fluid") or 150 ml/kg 24 h ("high fluid"). After 16 h of infection, rabbits that were given the lower fluid regimen had lower mean arterial blood pressure (MABP), lower CBF, and higher CSF lactate compared with animals that received the higher fluid regimen. In the first 4-6 h after antibiotic administration, low fluid rabbits had a significant decrease in MABP and CBF compared with, and a significantly greater increase in CSF lactate concentration than, high fluid rabbits. This study suggests that intravascular volume status may be a critical variable in determining CBF and therefore the degree of cerebral ischemia in meningitis.

Concurrent monitoring of cerebral electrophysiology and metabolism after traumatic brain injury: an experimental and clinical study

Multiparameter cerebral monitoring has been widely applied in traumatic brain injury to study posttraumatic pathophysiology and to manage head-injured patients (e.g., combining O(2) and pH sensors with cerebral microdialysis). Because a comprehensive approach towards understanding injury processes will also require functional measures, we have added electrophysiology to these monitoring modalities by attaching a recording electrode to the microdialysis probe. These dual-function (microdialysis/electrophysiology) probes were placed in rats following experimental fluid percussion brain injuries, and in a series of severely head-injured human patients. Electrical activity (cell firing, EEG) was monitored concurrently with microdialysis sampling of extracellular glutamate, glucose and lactate. Electrophysiological parameters (firing rate, serial correlation, field potential occurrences) were analyzed offline and compared to dialysate concentrations. In rats, these probes demonstrated an injury-induced suppression of neuronal firing (from a control level of 2.87 to 0.41 spikes/sec postinjury), which was associated with increases in extracellular glutamate and lactate, and decreases in glucose levels. When placed in human patients, the probes detected sparse and slowly firing cells (mean = 0.21 spike/sec), with most units (70%) exhibiting a lack of serial correlation in the spike train. In some patients, spontaneous field potentials were observed, suggesting synchronously firing neuronal populations. In both the experimental and clinical application, the addition of the recording electrode did not appreciably affect the performance of the microdialysis probe. The results suggest that this technique provides a functional monitoring capability which cannot be obtained when electrophysiology is measured with surface or epidural EEG alone.

Standard intracranial in vivo animal models of delayed cerebral vasospasm

Animal models provide a basis for clarifying the complex pathogenesis of delayed cerebral vasospasm (DCVS) and for screening of potential therapeutic approaches. Arbitrary use of experimental parameters in current models can lead to results of uncertain relevance. The aim of this work was to identify and analyze the most consistent and feasible models and their parameters for each animal.

Preliminary results of an ICP-controlled subarachnoid hemorrhage rabbit model for the study of delayed cerebral vasospasm

INTRODUCTION: Intracisternal blood injection is the most common applied experimental subarachnoid bleeding technique in rabbits. The model comprises examiner-dependent variables and does not closely represent the human pathophysiological sequelae of ruptured cerebral aneurysm. The degree of achieved delayed cerebral vasospasm (DCVS) in this model is often mild. The aim of this study was to characterize and evaluate the feasibility of a clinically more relevant experimental SAH in vivo model. SAH was performed by arterial blood shunting from the subclavian artery into the great cerebral cistern. A total of five experiments were performed. Intracranial pressure (ICP), arterial blood pressure, heart rate, arterial blood gas analysis, and neurological status were monitored throughout the experiments. SAH induced vasoconstriction of the basilar artery was 52.1±3.4% on day 3 compared to baseline (P<0.05). Post-mortem gross examination of the brain showed massive blood clot accumulation around the brainstem and ventral surface of the brain. The novel technique offers an examiner independent SAH induction and triggers high degrees of delayed cerebral vasospasm. The severity of vasospasm attained offers a unique opportunity to evaluate future therapeutic treatment options.

Structure-activity relationship and mechanism of action studies of manzamine analogues for the control of neuroinflammation and cerebral infections

Structure-activity relationship studies were carried out by chemical modification of manzamine A (1), 8-hydroxymanzamine A (2), manzamine F (14), and ircinal isolated from the sponge Acanthostrongylophora. The derived analogues were evaluated for antimalarial, antimicrobial, and antineuroinflammatory activities. Several modified products exhibited potent and improved in vitro antineuroinflammatory, antimicrobial, and antimalarial activity. 1 showed improved activity against malaria compared to chloroquine in both multi- and single-dose in vivo experiments. The significant antimalarial potential was revealed by a 100% cure rate of malaria in mice with one administration of 100 mg/kg of 1. The potent antineuroinflammatory activity of the manzamines will provide great benefit for the prevention and treatment of cerebral infections (e.g., Cryptococcus and Plasmodium). In addition, 1 was shown to permeate across the blood-brain barrier (BBB) in an in vitro model using a MDR-MDCK monolayer. Docking studies support that 2 binds to the ATP-noncompetitive pocket of glycogen synthesis kinase-3beta (GSK-3beta), which is a putative target of manzamines. On the basis of the results presented here, it will be possible to initiate rational drug design efforts around this natural product scaffold for the treatment of several different diseases.

Continuous intrathecal glyceryl trinitrate prevents delayed cerebral vasospasm in the single-SAH rabbit model in vivo

Delayed cerebral vasospasm after aneurysmal subarachnoid hemorrhage (SAH) is a major cause of high morbidity and mortality. The reduced availability of nitric oxide (NO) in blood and cerebrospinal fluid (CSF) is well established as a key mechanism of vasospasm. Systemic administration of glyceryl trinitrate (GTN), an NO donor also known as nitroglycerin, has failed to be established in clinical settings to prevent vasospasm because of its adverse effects, particularly hypotension. The purpose of this study was to analyze the effect of intrathecally administered GTN on vasospasm after experimental SAH in the rabbit basilar artery.

Activation of sphingosine kinase 2 is an endogenous protective mechanism in cerebral ischemia

The two ubiquitously expressed sphingosine kinases (SphK) 1 and 2 are key regulators of the sphingolipid signaling pathway. Despite the formation of an identical messenger, i.e. sphingosine 1-phosphate (S1P), they exert strikingly different functions. Particularly, SphK2 is necessary for the phosphorylation of the sphingosine analog fingolimod (FTY720), which is protective in rodent stroke models. Using gene deficient mice lacking either SphK1 or SphK2, we investigated the role of the two lipid kinases in experimental stroke. We performed 2h transient middle cerebral artery occlusion (tMCAO) and analyzed lesion size and neurological function after 24h. Treatment groups received 1mg/kg FTY720. Neutrophil infiltration, microglia activation, mRNA and protein expression of SphK1, SphK2 and the S1P(1) receptor after tMCAO were studied. Genetic deletion of SphK2 but not SphK1 increased ischemic lesion size and worsened neurological function after tMCAO. The protective effect of FTY720 was conserved in SphK1(-/-) mice but not in SphK2(-/-) mice. This suggests that SphK2 activity is an important endogenous protective mechanism in cerebral ischemia and corroborates that the protective effect of FTY720 is mediated via phospho-FTY720.

Outer skull landmark-based coordinates for measurement of cerebral blood flow and intracranial pressure in rabbits

Despite the increased use of intracranial neuromonitoring during experimental subarachnoid hemorrhage (SAH), coordinates for probe placement in rabbits are lacking. This study evaluates the safety and reliability of using outer skull landmarks to identify locations for placement of cerebral blood flow (CBF) and intraparenchymal intracranial pressure (ICP) probes. Experimental SAH was performed in 17 rabbits using an extracranial-intracranial shunt model. ICP probes were placed in the frontal lobe and compared to measurements recorded from the olfactory bulb. CBF probes were placed in various locations in the frontal cortex anterior to the coronary suture. Insertion depth, relation to the ventricular system, and ideal placement location were determined by post-mortem examination. ICP recordings at the time of SAH from the frontal lobe did not differ significantly from those obtained from the right olfactory bulb. Ideal coordinates for intraparenchymal CBF probes in the left and right frontal lobe were found to be located 4.6±0.9 and 4.5±1.2 anterior to the bregma, 4.7±0.7mm and 4.7±0.5mm parasagittal, and at depths of 4±0.5mm and 3.9±0.5mm, respectively. The results demonstrate that the presented coordinates based on skull landmarks allow reliable placement of intraparenchymal ICP and CBF probes in rabbit brains without the use of a stereotactic frame.

3D Computerized Occlusion Rating of Embolized Experimental Aneurysms Using Noninvasive 1.5T MR Imaging

For embolized cerebral aneurysms, the initial occlusion rate is the most powerful parameter to predict aneurysm rerupture and recanalization. However, the occlusion rate is only estimated subjectively in clinical routine. To minimize subjective bias, computer occlusion-rating (COR) was successfully validated for 2D images. To minimize the remaining inaccuracy of 2D-COR, COR was applied to 1.5T 3D MR imaging.

Experimental subarachnoid hemorrhage causes early and long-lasting microarterial constriction and microthrombosis: an in-vivo microscopy study

Early brain injury (EBI) after subarachnoid hemorrhage (SAH) is characterized by a severe, cerebral perfusion pressure (CPP)-independent reduction in cerebral blood flow suggesting alterations on the level of cerebral microvessels. Therefore, we aimed to use in-vivo imaging to investigate the cerebral microcirculation after experimental SAH. Subarachnoid hemorrhage was induced in C57/BL6 mice by endovascular perforation. Pial arterioles and venules (10 to 80 μm diameter) were examined using in-vivo fluorescence microscopy, 3, 6, and 72 hours after SAH. Venular diameter or flow was not affected by SAH, while >70% of arterioles constricted by 22% to 33% up to 3 days after hemorrhage (P<0.05 versus sham). The smaller the investigated arterioles, the more pronounced the constriction (r(2)=0.92, P<0.04). Approximately 30% of constricted arterioles were occluded by microthrombi and the frequency of arteriolar microthrombosis correlated with the degree of constriction (r(2)=0.93, P<0.03). The current study demonstrates that SAH induces microarterial constrictions and microthrombosis in vivo. These findings may explain the early CPP-independent decrease in cerebral blood flow after SAH and may therefore serve as novel targets for the treatment of early perfusion deficits after SAH.

RecNcMIC3-1-R is a microneme- and rhoptry-based chimeric antigen that protects against acute neosporosis and limits cerebral parasite load in the mouse model for Neospora caninum infection

In order to achieve host cell entry, the apicomplexan parasite Neospora caninum relies on the contents of distinct organelles, named micronemes, rhoptries and dense granules, which are secreted at defined timepoints during and after host cell entry. It was shown previously that a vaccine composed of a mixture of three recombinant antigens, corresponding to the two microneme antigens NcMIC1 and NcMIC3 and the rhoptry protein NcROP2, prevented disease and limited cerebral infection and transplacental transmission in mice. In this study, we selected predicted immunogenic domains of each of these proteins and created four different chimeric antigens, with the respective domains incorporated into these chimers in different orders. Following vaccination, mice were challenged intraperitoneally with 2 × 10(6)N. caninum tachzyoites and were then carefully monitored for clinical symptoms during 4 weeks post-infection. Of the four chimeric antigens, only recNcMIC3-1-R provided complete protection against disease with 100% survivors, compared to 40-80% of survivors in the other groups. Serology did not show any clear differences in total IgG, IgG1 and IgG2a levels between the different treatment groups. Vaccination with all four chimeric variants generated an IL-4 biased cytokine expression, which then shifted to an IFN-γ-dominated response following experimental infection. Sera of recNcMIC3-1-R vaccinated mice reacted with each individual recombinant antigen, as well as with three distinct bands in Neospora extracts with similar Mr as NcMIC1, NcMIC3 and NcROP2, and exhibited distinct apical labeling in tachyzoites. These results suggest that recNcMIC3-1-R is an interesting chimeric vaccine candidate and should be followed up in subsequent studies in a fetal infection model.

Experimental treatment of Neospora caninum-infected mice with the arylimidamide DB750 and the thiazolide nitazoxanide

The cationic arylimidamide DB750 and the thiazolide nitazoxanide had been shown earlier to be effective against Neospora caninum tachyzoites in vitro with an IC(50) of 160nM and 4.23muM, respectively. In this study, we have investigated the effects of DB750 and nitazoxanide treatments of experimentally infected Balb/c mice, by applying the drugs either through the oral or the intraperitoneal route. In experiment 1, administration of DB750 (2mg/kg/day) and nitazoxanide (150mg/kg/day) started already 3 days prior to experimental infection of mice with 2x10(6) tachyzoites. Following infection, the drugs were further administrated daily for a period of 2 weeks, either orally or intraperitoneally. Intraperitoneal injection of DB750 was well tolerated by the mice, but treatment with nitazoxanide resulted in death of all mice within 3 days. Upon intraperitoneal application of DB750, the cerebral parasite load was significantly reduced compared to all other groups, while oral application of DB750 and nitazoxanide were not as effective, and resulted in significant weight loss. In experiment 2, mice were infected with 2x10(6) tachyzoites and at 2 weeks post-infection, DB750 (2mg/kg/day) was applied by intraperitoneal injections for 14 days. In the DB750-treated group, only 2 out of 12 mice succumbed to infection, compared to 7 out of 12 mice in the placebo-group. DB750 treatment also resulted in significantly reduced cerebral parasite burden, and reduced numbers of viable tachyzoites. Our data suggest that DB750 exerted its activity also after crossing the blood-brain barrier, and that this class of compounds could be promising for the control of N. caninum-associated disease.