Patricia Alves Reis

Cognitive Dysfunction Is Sustained after Rescue Therapy in Experimental Cerebral Malaria, and Is Reduced by Additive Antioxidant Therapy

Neurological impairments are frequently detected in children surviving cerebral malaria (CM), the most severe neurological complication of infection with Plasmodium falciparum. The pathophysiology and therapy of long lasting cognitive deficits in malaria patients after treatment of the parasitic disease is a critical area of investigation. In the present study we used several models of experimental malaria with differential features to investigate persistent cognitive damage after rescue treatment. Infection of C57BL/6 and Swiss (SW) mice with Plasmodium berghei ANKA (PbA) or a lethal strain of Plasmodium yoelii XL (PyXL), respectively, resulted in documented CM and sustained persistent cognitive damage detected by a battery of behavioral tests after cure of the acute parasitic disease with chloroquine therapy. Strikingly, cognitive impairment was still present 30 days after the initial infection. In contrast, BALB/c mice infected with PbA, C57BL6 infected with Plasmodium chabaudi chabaudi and SW infected with non lethal Plasmodium yoelii NXL (PyNXL) did not develop signs of CM, were cured of the acute parasitic infection by chloroquine, and showed no persistent cognitive impairment. Reactive oxygen species have been reported to mediate neurological injury in CM. Increased production of malondialdehyde (MDA) and conjugated dienes was detected in the brains of PbA-infected C57BL/6 mice with CM, indicating high oxidative stress. Treatment of PbA-infected C57BL/6 mice with additive antioxidants together with chloroquine at the first signs of CM prevented the development of persistent cognitive damage. These studies provide new insights into the natural history of cognitive dysfunction after rescue therapy for CM that may have clinical relevance, and may also be relevant to cerebral sequelae of sepsis and other disorders.

Statins decrease neuroinflammation and prevent cognitive impairment after cerebral malaria

Cerebral malaria (CM) is the most severe manifestation of Plasmodium falciparum infection in children and non-immune adults. Previous work has documented a persistent cognitive impairment in children who survive an episode of CM that is mimicked in animal models of the disease. Potential therapeutic interventions for this complication have not been investigated, and are urgently needed. HMG-CoA reductase inhibitors (statins) are widely prescribed for cardiovascular diseases. In addition to their effects on the inhibition of cholesterol synthesis, statins have pleiotropic immunomodulatory activities. Here we tested if statins would prevent cognitive impairment in a murine model of cerebral malaria. Six days after infection with Plasmodium berghei ANKA (PbA) mice displayed clear signs of CM and were treated with chloroquine, or chloroquine and lovastatin. Intravital examination of pial vessels of infected animals demonstrated a decrease in functional capillary density and an increase in rolling and adhesion of leukocytes to inflamed endothelium that were reversed by treatment with lovastatin. In addition, oedema, ICAM-1, and CD11b mRNA levels were reduced in lovastatin-treated PbA-infected mice brains. Moreover, HMOX-1 mRNA levels are enhanced in lovastatin-treated healthy and infected brains. Oxidative stress and key inflammatory chemokines and cytokines were reduced to non-infected control levels in animals treated with lovastatin. Fifteen days post-infection cognitive dysfunction was detected by a battery of cognition tests in animals rescued from CM by chloroquine treatment. In contrast, it was absent in animals treated with lovastatin and chloroquine. The outcome was similar in experimental bacterial sepsis, suggesting that statins have neuroprotective effects in severe infectious syndromes in addition to CM. Statin treatment prevents neuroinflammation and blood brain barrier dysfunction in experimental CM and related conditions that are associated with cognitive sequelae, and may be a valuable adjuvant therapeutic agent for prevention of cognitive impairment in patients surviving an episode of CM.

Blockade of the renin–angiotensin system improves cerebral microcirculatory perfusion in diabetic hypertensive rats☆

We examined the functional and structural microcirculatory alterations in the brain, skeletal muscle and myocardium of non-diabetic spontaneously hypertensive rats (SHR) and diabetic SHR (D-SHR), as well as the effects of long-term treatment with the angiotensin AT1-receptor antagonist olmesartan and the angiotensin-converting enzyme inhibitor enalapril. Diabetes was experimentally induced by a combination of a high-fat diet with a single low dose of streptozotocin (35 mg/kg, intraperitoneal injection). D-SHR were orally administered with olmesartan (5 mg/kg/day), enalapril (10 mg/kg/day) or vehicle for 28 days, and compared with vehicle-treated non-diabetic SHR or normotensive non-diabetic Wistar-Kyoto rats. The cerebral and skeletal muscle functional capillary density of pentobarbital-anesthetized rats was assessed using intravital fluorescence videomicroscopy. Chronic treatment with olmesartan or enalapril significantly lowered blood pressure and reversed brain functional capillary rarefaction. Brain oxidative stress was reduced to non-diabetic control levels in animals treated with olmesartan or enalapril. Histochemical analysis of the structural capillary density showed that both olmesartan and enalapril increased the capillary-to-fiber ratio in skeletal muscle and the capillary-to-fiber volume density in the left ventricle. Olmesartan and enalapril also prevented collagen deposition and the increase in cardiomyocyte diameter in the left ventricle. Our results suggest that the association between hypertension and diabetes results in microvascular alterations in the brain, skeletal muscle and myocardium that can be prevented by chronic blockade of the renin-angiotensin system.

Sofosbuvir protects Zika virus-infected mice from mortality, preventing short- and long-term sequelae

Zika virus (ZIKV) causes significant public health concerns because of its association with congenital malformations, neurological disorders in adults, and, more recently, death. Considering the necessity to mitigate ZIKV-associated diseases, antiviral interventions are an urgent necessity. Sofosbuvir, a drug in clinical use against hepatitis C virus (HCV), is among the FDA-approved substances endowed with anti-ZIKV activity. In this work, we further investigated the in vivo activity of sofosbuvir against ZIKV. Neonatal Swiss mice were infected with ZIKV (2 × 107 PFU) and treated with sofosbuvir at 20 mg/kg/day, a concentration compatible with pre-clinical development of this drug. We found that sofosbuvir reduced acute levels of ZIKV from 60 to 90% in different anatomical compartments, such as the blood plasma, spleen, kidney, and brain. Early treatment with sofosbuvir doubled the percentage and time of survival of ZIKV-infected animals. Sofosbuvir also prevented the acute neuromotor impairment triggered by ZIKV. In the long-term behavioural analysis of ZIKV-associated sequelae, sofosbuvir prevented loss of hippocampal- and amygdala-dependent memory. Our results indicate that sofosbuvir inhibits ZIKV replication in vivo, which is consistent with the prospective necessity of antiviral drugs to treat ZIKV-infected individuals.

Effects of experimental cerebral malaria in memory, brain-derived neurotrophic factor and acetylcholinesterase acitivity in the hippocampus of survivor mice

Malaria is the most important human parasitic disease and cerebral malaria (CM), its main neurological complication, is characterized by neurological and cognitive damage in both human and animal survivors. The brain-derived neurotrophic factor (BDNF) appears to be involved with activity-dependent synaptic plasticity. There is great interest regarding its role in learning and memory as well as acetylcholinesterase activity (AChE) that is implicated in many cognitive functions and probably plays important roles in neurodegenerative disorders. In the present work, we evaluated BDNF protein levels and AChE activity in the hippocampus and habituation in an animal model of CM using C57BL/6 mice after fifteen days of the induction. The results demonstrated that there was a decrease in BDNF levels in the hippocampus of C57BL/6 mice infected with PbA when compared with C57BL/6 non-infected mice and C57BL/6 non-infected mice that received treatment with chloroquine. However, no difference was observed in AChE activity in the hippocampus. When habituation was evaluated there was memory impairment in the C57BL/6 mice infected with Plasmodium berghei ANKA (PbA). In conclusion, we believe that the decreased BDNF levels in the hippocampus may be related with memory impairment without alterations on AChE activity.

Statins prevent cognitive impairment after sepsis by reverting neuroinflammation, and microcirculatory/endothelial dysfunction

Acute brain dysfunction is a frequent condition in sepsis patients and is associated with increased mortality and long-term neurocognitive consequences. Impaired memory and executive function are common findings in sepsis survivors. Although neuroinflammation and blood-brain barrier dysfunction have been associated with acute brain dysfunction and its consequences, no specific treatments are available that prevent cognitive impairment after sepsis. Experimental sepsis was induced in Swiss Webster mice by intraperitoneal injection of cecal material (5mg/kg, 500μL). Control groups (n=5/group each experiment) received 500μL of saline. Support therapy recover (saline 0.9%, 1mL and imipenem 30mg/kg) were applied (6, 24 and 48h post injection, n=5-10/group, each experiment), together or not with additive orally treatment with statins (atorvastatin/simvastatin 20mg/kg b.w.). Survival rate was monitored at 6, 24 and 48h. In a setting of experiments, animals were euthanized at 6 and 24h after induction for biochemical, immunohistochemistry and intravital analysis. Statins did not prevented mortality in septic mice, however survivors presented lower clinical score. At another setting of experiments, after 15days, mice survivors from fecal supernatant peritoneal sepsis presented cognitive dysfunction for contextual hippocampal and aversive amygdala-dependent memories, which was prevented by atorvastatin/simvastatin treatment. Systemic and brain tissue levels of proinflammatory cytokines/chemokines and activation of microglial were lower in septic mice treated with statins. Brain lipid peroxidation and myeloperoxidase levels were also reduced by statins treatment. Intravital examination of the brain vessels of septic animals revealed decreased functional capillary density and increased rolling and adhesion of leukocytes, and blood flow impairment, which were reversed by treatment with statins. In addition, treatment with statins restored the cholinergic vasodilator response due to sepsis. Taken together, these data demonstrated that statins reverse microvascular dysfunction and reduce neuroinflammation during sepsis, preventing the development of long-term cognitive decline.

Acute Chagas Disease Induces Cerebral Microvasculopathy in Mice

Cardiomyopathy is the main clinical form of Chagas disease (CD); however, cerebral manifestations, such as meningoencephalitis, ischemic stroke and cognitive impairment, can also occur. The aim of the present study was to investigate functional microvascular alterations and oxidative stress in the brain of mice in acute CD. Acute CD was induced in Swiss Webster mice (SWM) with the Y strain of Trypanosoma cruzi (T. cruzi). Cerebral functional capillary density (the number of spontaneously perfused capillaries), leukocyte rolling and adhesion and the microvascular endothelial-dependent response were analyzed over a period of fifteen days using intravital video-microscopy. We also evaluated cerebral oxidative stress with the thiobarbituric acid reactive species TBARS method. Compared with the non-infected group, acute CD significantly induced cerebral functional microvascular alterations, including (i) functional capillary rarefaction, (ii) increased leukocyte rolling and adhesion, (iii) the formation of microvascular platelet-leukocyte aggregates, and (iv) alteration of the endothelial response to acetylcholine. Moreover, cerebral oxidative stress increased in infected animals. We concluded that acute CD in mice induced cerebral microvasculopathy, characterized by a reduced incidence of perfused capillaries, a high number of microvascular platelet-leukocyte aggregates, a marked increase in leukocyte-endothelium interactions and brain arteriolar endothelial dysfunction associated with oxidative stress. These results suggest the involvement of cerebral microcirculation alterations in the neurological manifestations of CD.

Combined therapy with metformin and insulin attenuates systemic and hepatic alterations in a model of high-fat diet-/streptozotocin-induced diabetes

In this study we have explored the pathogenesis of the hepatic alterations which occur in diabetes and the modulation of these complications by the combination of metformin adjunct treatment and insulin monotherapy. For this purpose, diabetic rats were treated with insulin (DM + Ins) or metformin plus insulin (DM + Met + Ins). Biochemical and cardiometabolic parameters were analysed by spectrophotometry. Intravital microscopy was used to study the hepatic microcirculation. In the liver tissue, real‐time PCR was used to analyse oxidative stress enzymes, inflammatory markers and receptors for advanced glycation end products (AGE) (RAGE) gene expression. Lipid peroxidation was assessed by thiobarbituric acid reactive species (TBARs) analyses. AGE deposition and RAGE protein expression were studied by fluorescence spectrophotometry and Western blot respectively. Body weight, %HbA1c, urea, total proteins and oxidative stress parameters were found to be similarly improved by insulin or Met + Ins treatments. On the other hand, Met + Ins treatment showed a more pronounced effect on fasting blood glucose level than insulin monotherapy. Fructosamine, uric acid, creatinine, albumin and amylase levels and daily insulin dose requirements were found to be only improved by the combined Met + Ins treatment. Liver, renal and pancreatic dysfunction markers were found to be more positively affected by metformin adjunct therapy when compared to insulin treatment. Liver microcirculation damage was found to be completely protected by Met + Ins treatment, while insulin monotherapy showed no effect. Our results suggest that oxidative stress, microcirculatory damage and glycated proteins could be involved in the aetiology of liver disease due to diabetes. Additionally, metformin adjunct treatment improved systemic and liver injury in induced diabetes and showed a more pronounced effect than insulin monotherapy.

Acute simvastatin treatment restores cerebral functional capillary density and attenuates angiotensin II-induced microcirculatory changes in a model of primary hypertension

We investigated the acute effects of SIM on cerebral microvascular rarefaction and dysfunction in SHRs.

Yellow fever virus is susceptible to sofosbuvir both in vitro and in vivo

Yellow fever virus (YFV) is a member of the Flaviviridae family, that causes major mortality. In Brazil, YFV activity increased in the last years. It has been registered that sylvatic, instead of urban, yellow fever (YF) leads our contemporary public health concern. Low vaccinal coverage leaves the human population near the jangle vulnerable to the outbreak, making it necessary to identify therapeutic options. Repurposing of clinically approved antiviral drugs represents an alternative for such identification. Other Flaviviruses, such Zika (ZIKV) and dengue (DENV) viruses, are susceptible to Sofosbuvir, a clinically approved drug against hepatitis C virus (HCV). Moreover, sofosbuvir has a safety record on critically ill hepatic patients, making it an attractive option. Our data show that YFV RNA polymerase uses conserved amino acid resides for nucleotide binding to dock sofosbuvir. This drug inhibited YFV replication in different lineages of human hepatoma cells, Huh-7 and HepG2, with EC50 value of 4.8 µM. Sofosbuvir protected YFV-infected neonatal Swiss mice from mortality and weight loss. Our pre-clinical results indicate that sofosbuvir could represent an option against YFV.