Mitochondrial pyruvate carrier as a key regulator of fever and neuroinflammation.

The mitochondrial pyruvate carrier (MPC) is an inner-membrane transporter that facilitates pyruvate uptake from the cytoplasm into mitochondria. We previously reported that MPC1 protein levels increase in the hypothalamus of animals during fever induced by lipopolysaccharide (LPS), but how this increase contributes to the LPS responses remains to be studied. Therefore, we investigated the effect of UK 5099, a classical MPC inhibitor, in a rat model of fever, on hypothalamic mitochondrial function and neuroinflammation in LPS-stimulated preoptic area (POA) primary microcultures. Intracerebroventricular administration of UK 5099 reduced the LPS-induced fever. High-resolution respirometry revealed an increase in oxygen consumption and oxygen flux related to ATP synthesis in the hypothalamic homogenate from LPS-treated animals linked to mitochondrial complex I plus II. Preincubation with UK 5099 prevented the LPS-induced increase in oxygen consumption, ATP synthesis and spare capacity only in complex I-linked respiration and reduced mitochondrial H2O2 production. In addition, treatment of rat POA microcultures with UK 5099 reduced the secretion of the proinflammatory and pyrogenic cytokines TNFα and IL-6 as well as the immunoreactivity of inflammatory transcription factors NF-κB and NF-IL6 four hours after LPS stimulation. These results suggest that the regulation of mitochondrial pyruvate metabolism through MPC inhibition may be effective in reducing neuroinflammation and fever.

In Vivo Evaluation of DMSA-Coated Magnetic Nanoparticle Toxicity and Biodistribution in Rats: A Long-Term Follow-Up.

This work presents a long-term follow-up (300 days) of rats after a single intravenous injection of DMSA-coated magnetite nanoparticles (DMSA-MNP). The animals were systematically evaluated by hematological, biochemical, and ultrasound examinations, monitoring the same animal over time. In addition, oxidative stress evaluation, DMSA-MNP biodistribution, computerized tomography for ex vivo organs, and histopathology analysis were performed at the end of the experiment period. Overall, DMSA-MNP administration did not cause serious damage to the rats' health over the course of 300 days post-administration. All animals presented hematological parameters within the normal limits, and no alterations on serum creatinine, urea, ALT, and AST were related to DMSA-MNP administration. Liver and spleen showed no important alterations in any of the examinations. The kidneys of treated animals displayed intermittent pelvis dilation at ultrasound analysis, but without damage to the organ parenchyma after 300 days. The lungs of treated animals presented a light interalveolar septal thickening, but the animals did not present any clinical respiratory symptom. Nanoparticles were not detected in the vital organs of treated animals 300 days after administration. This work represents the first assessment of the long-term effects of DMSA-MNP and goes a step further on the safety of its use for biomedical applications.

Evaluation of Biocompatibility, Anti-Inflammatory, and Antinociceptive Activities of Pequi Oil-Based Nanoemulsions in In Vitro and In Vivo Models.

Pequi oil (Caryocar brasiliense) contains bioactive compounds capable of modulating the inflammatory process; however, its hydrophobic characteristic limits its therapeutic use. The encapsulation of pequi oil in nanoemulsions can improve its biodistribution and promote its immunomodulatory effects. Thus, the objective of the present study was to formulate pequi oil-based nanoemulsions (PeNE) to evaluate their biocompatibility, anti-inflammatory, and antinociceptive effects in in vitro (macrophages­J774.16) and in vivo (Rattus novergicus) models. PeNE were biocompatible, showed no cytotoxic and genotoxic effects and no changes in body weight, biochemistry, or histology of treated animals at all concentrations tested (90−360 µg/mL for 24 h, in vitro; 100−400 mg/kg p.o. 15 days, in vivo). It was possible to observe antinociceptive effects in a dose-dependent manner in the animals treated with PeNE, with a reduction of 27 and 40% in the doses of 100 and 400 mg/kg of PeNE, respectively (p < 0.05); however, the treatment with PeNE did not induce edema reduction in animals with carrageenan-induced edema. Thus, the promising results of this study point to the use of free and nanostructured pequi oil as a possible future approach to a preventive/therapeutic complementary treatment alongside existing conventional therapies for analgesia.

Luminescent Marker for GSR: Evaluation of the Acute Oral and Inhalation Toxicity of the MOF [Eu(DPA)(HDPA)].

The metal-organic framework (MOF) [Eu(DPA)(HDPA)] (where DPA is dipicolinic acid) has been previously reported as an efficient marker for gunshot residues (GSRs). Since this marker will be in contact with various shooters, industrial workers, and the environment, however, it is important to identify its toxicity. In this work, the oral and the inhalation acute toxicities of the MOF [Eu(DPA)(HDPA)] (also called R-Marker) were evaluated in young Wistar rats using Guidelines 423 (oral) and 436 (inhalation) from the Organisation for Economic Co-operation and Development (OECD). Animal behavior; body weight, water, and food intake; and organ weight, as well as biochemical parameters were evaluated in both evaluations. For the inhalation test, a concentration of 1 mg·Lair-1·(4 h-1) was reached in a whole-body inhalation chamber. When the respiratory tract was analyzed, it was observed that part of the marker had been swallowed instead of inhaled by the animal. For the oral test, the highest administrated dose was 2000 mg/kg with no sign of toxicity. This marker has been classified in the least toxic category of the Globally Harmonized System (GHS; category 5), with an oral median lethal dose (LD50) of 5000 mg/kg. After the oral administration, the feces of the animals were collected using a metabolic cage. Luminescent feces were present up to 24 h after administration, indicating that the marker had been excreted by the organism without causing intoxication. This study has opened perspectives for drug delivery and toxicity studies, since it enables visual detection of the marker.

Cytoglobin Attenuates Neuroinflammation in Lipopolysaccharide-Activated Primary Preoptic Area Cells via NF-κB Pathway Inhibition.

Cytoglobin (Cygb) is a hexacoordinate protein, associated with the transport of oxygen, nitric oxide scavenging, tumor suppression and protection against oxidative stress and inflammation. This protein is expressed in brain areas including the preoptic area (POA) of the anterior hypothalamus, the region responsible for the regulation of body temperature. In this study, we show that Cygb is upregulated in the rat hypothalamus 2.5 h and 5 h after intravenous administration of lipopolysaccharide (LPS). We investigated the effect of treatment with Cygb in POA primary cultures stimulated with LPS for 4 h. The levels of tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) were measured and the results showed that Cygb reduced the concentrations of both cytokines. We further observed a decrease in immunoreactivity of the inflammatory transcription factor nuclear factor-κB (NF-κB), but not NF-IL6 and STAT3, in the nucleus of Cygb-treated POA cells. These findings suggest that Cygb attenuates the secretion of IL-6 and TNF-α in LPS-stimulated POA primary cultures via inhibition of the NF-κB signaling pathway, indicating that this protein might play an important role in the control of neuroinflammation and fever.

Combination of cyclodextrin complexation and iontophoresis as a promising strategy for the cutaneous delivery of aluminum-chloride phthalocyanine in photodynamic therapy.

Topical application of aluminum-chloride phthalocyanine (AlClPc) is a challenge because of the drug's extremely low solubility, which prevents its absorption into deeper skin layers and causes molecule aggregation, reducing the photophysical effect. The goal of this study was to obtain a formulation applied in a certain condition that would allow homogeneous accumulation of AlClPc in cutaneous tissues, meaning a safer and non-invasive topical treatment for skin tumors based on photodynamic therapy. We first prepared and characterized AlClPc complexes with cyclodextrin to increase the photosensitizing agent solubility. The inclusion complex of AlClPc with hydroxypropyl-ß-cyclodextrin (HP-ßCD) amplified its loading dose in aqueous medium and maintained its photosensitizing properties in terms of reactive oxygen species production. Assays to determine the complex's in vitro cytotoxicity against murine melanoma skin cancer cells showed that when irradiated, the complex significantly reduced cell viability, whereas the absence of irradiation did not affect cell viability. Three physical techniques for permeation enhancement (i.e., tape-stripping abrasion, microneedle pretreatment and iontophoresis) were then evaluated. When applied in impaired skin, the complex could not increase drug penetration. The skin penetration of AlClPc, however, increased 2.3-fold following iontophoresis application in a shorter period compared to passive permeation. Therefore, these results suggest the administration of complexed AlClPc mediated by iontophoresis, followed by application of photodynamic therapy, might be an effective and non-invasive alternative for topical treatment of cutaneous tumors.

Increase of reactive oxygen species in different tissues during lipopolysaccharide-induced fever and antipyresis: an electron paramagnetic resonance study.

Fever is a regulated increase in body temperature and a component of the acute-phase response, triggered mainly after the invasion of pathogens in the body. Reactive oxygen species (ROS) are generated during the physiological and pathological processes, and can act as both signalling molecules as well as promoters of oxidative stress. Male Wistar rats, pretreated with oral doses of acetaminophen, celecoxib, dipyrone, or ibuprofen 30 min before an intravenous lipopolysaccharide (LPS) or sterile saline injection, showed a reduced febrile response in all animals tested. The formation of ROS in the fresh blood, liver, brown adipose tissue (BAT), and hypothalamus of febrile and antipyretic-treated animals was assessed by electron paramagnetic resonance using the spin probe 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH). While the CM• concentrations remained unaltered in the blood samples examined 5 h after the induction of fever, we found increased CM• levels in the liver (in µM, saline: 290 ± 42; LPS: 512 ± 34), BAT (in µM, saline: 509 ± 79, LPS: 855 ± 79), and hypothalamus (in µM, saline: 292 ± 35; LPS: 467 ± 8) at the same time point. Importantly, none of the antipyretics were seen to alter the CM• accumulation profile. Data from this study suggest that there is an increased formation of ROS in the different tissues during fever, which may cause oxidative stress, and that the antipyretics tested do not interfere with ROS production.

Label-free quantitative proteomics of rat hypothalamus under fever induced by LPS and PGE2.

Fever is a brain-mediated increase in body temperature mainly during inflammatory or infectious challenges. Although there is considerable data regarding the inflammation pathways involved in fever, metabolic alterations necessary to orchestrate the complex inflammatory response are not totally understood. We performed proteomic analysis of rat hypothalamus using label-free LC-MS/MS in a model of fever induced by lipopolysaccharide (LPS) or prostaglandin E2 (PGE2). In total, 7021 proteins were identified. As far as we know, this is the largest rat hypothalamus proteome dataset available to date. Pathway analysis showed proteins from both stimuli associated with inflammatory and metabolic pathways. Concerning metabolic pathways, rats exposed to LPS or PGE2 presented lower relative abundance of proteins involved in glycolysis, pentose phosphate pathway and tricarboxylic acid cycle. Mitochondrial function may also be altered by both stimuli because significant downregulation of several proteins was found, mainly in complexes I and IV. LPS was able to induce downregulation of important proteins in the enzymatic antioxidant system, thereby contributing to oxidative stress. The results offered comprehensive information about fever responses and helped to reveal new insights into proteins potentially involved in inflammatory signaling and metabolic changes in the hypothalamus during systemic LPS and central PGE2 administration. SIGNIFICANCE: The evolutionary persistence of fever, despite the elevated cost for maintenance of this response, suggests that elevation in core temperature may represent an interesting strategy for survival. Fever response is achieved through the integrated behavioral, physiological, immunological and biochemical processes that determine the balance between heat generation and elimination. The development of such complex response arouses interest in studying how the cell metabolism responds or even contributes to promote fever. Our results offered comprehensive information about fever responses, including metabolic and inflammatory pathways, providing new insights into candidate proteins potentially involved in inflammatory signaling and metabolic changes in the hypothalamus during fever induced by systemic LPS and central PGE2 perturbation.

Application of the Metal-Organic Framework [Eu(BTC)] as a Luminescent Marker for Gunshot Residues: A Synthesis, Characterization, and Toxicity Study.

The 3D metal-organic framework (MOF) [Eu(BTC)] (where BTC = trimesic acid) was synthesized in 20 min by a microwave-assisted hydrothermal method with a yield of 89%. A structural and spectroscopic study, performed by X-ray diffraction, thermogravimetry, and photoluminescence spectroscopy, showed that this framework has high crystallinity, thermal stability, and luminescence. This MOF had a red-orange luminescence when excited with ultraviolet (UV) radiation (λ = 254 nm) and a high potential for use as a luminescent marker for gunshot residues (GSR). When added to 9 mm nontoxic ammunition, it greatly improved quality of the crime scene investigation, allowing for direct visualization of the luminescent GSR on the shooter's hand and firearm and at the firing range using only a portable UV lamp. The marked luminescent GSR was easily collected and characterized by nondestructive techniques, including with a Video Spectral Comparator and scanning electron microscopy/energy-dispersive spectroscopy, wherein the presence of Eu3+ ions was confirmed. Furthermore, the oral acute toxicity of this MOF was assessed in adult female Wistar rats using the Organisation for Economic Cooperation and Development 423 guidelines. This study classified the MOF [Eu(BTC)] in a less toxic Globally Harmonized System category (category 5), with a LD50 (lethal dose) of 5000 mg/kg, ensuring a wide security range for its application.

Cyclooxygenase-independent mechanism of ibuprofen-induced antipyresis: the role of central vasopressin V1 receptors.

This study compared the antipyretic effects of ibuprofen and indomethacin regarding the efficacy in blocking fevers induced by lipopolysaccharide (LPS from E. coli) or pyrogenic mediators that act on prostaglandin (PG)-dependent and PG-independent pathways. The content of PGE2 in the cerebrospinal fluid (CSF) and the dependence on central arginine vasopressin (AVP) release by both antipyretics were also compared during the reduction of LPS-induced fever. Finally, we investigated the effect of ibuprofen on hypothalamic cytokine content during LPS-induced fever. Ibuprofen (intraperitoneally, i.p.) dose-dependently inhibited the fever induced by LPS (intravenously, i.v.). Indomethacin (2 mg/kg) and ibuprofen (10 mg/kg) reduced the fever induced by i.c.v. injection of interleukin (IL)-1ß, IL-6, tumor necrosis factor (TNF)-α, or arachidonic acid (AA). Ibuprofen, but not indomethacin, inhibited i.c.v. endothelin-1- and pre-formed pyrogenic factor (PFPF)-induced fever. Neither ibuprofen nor indomethacin affected fever by PGE2 , PGF(2α) , or corticotrophin-releasing factor (CRF); however, both reduced the CSF PGE2 content after LPS. Bilateral injection of the AVP V(1) receptor antagonist d(CH2)5 Tyr(Me)AVP into the ventral septal area blocked both ibuprofen- and indomethacin-induced antipyresis. Ibuprofen did not modify the hypothalamic increase in either IL-1ß or IL-6 induced by LPS. In conclusion, although the antipyretic effect of ibuprofen involves the blockage of central production of PGE2 and the endogenous release of AVP, differently from low dose of indomethacin, ibuprofen not only reduced the fever induced by PGE2 -dependent, but also, that induced by PGE2 -independent endogenous pyrogens. Moreover, ibuprofen does not affect the hypothalamic synthesis/release of IL-1ß and IL-6.