Melatonin renders neuroprotection by protein kinase C mediated aquaporin-4 inhibition in animal model of focal cerebral ischemia

Aim

Aquaporin-4(AQP4) expression in the brain with relation to edema formation following focal cerebral ischemia was investigated. Studies have shown that brain edema is one of the significant factors in worsening stroke outcomes. While many mechanisms may aggravate brain injury, one such potential system may involve AQP4 up regulation in stroke patients that could result in increased edema formation. Post administration of melatonin following ischemic stroke reduces AQP4 mediated brain edema and confers neuroprotection.

Materials and methods

An in-silico approach was undertaken to confirm effective melatonin-AQP4 binding. Rats were treated with 5 mg/kg, i.p. melatonin or placebo at 30 min prior, 60 min post and 120 min post 60 min of middle cerebral artery occlusion (MCAO) followed by 24 h reperfusion. Rats were evaluated for battery of neurological and motor function tests just before sacrifice. Brains were harvested for infarct size estimation, water content measurement, biochemical analysis, apoptosis study and western blot experiments.

Key findings

Melatonin at 60 min post ischemia rendered neuroprotection as evident by reduction in cerebral infarct volume, improvement in motor and neurological deficit and reduction in brain edema. Furthermore, ischemia induced surge in levels of nitrite and malondialdehyde (MDA) were also found to be significantly reduced in ischemic brain regions in treated animals. Melatonin potentiated intrinsic antioxidant status, inhibited acid mediated rise in intracellular calcium levels, decreased apoptotic cell death and also markedly inhibited protein kinase C (PKC) influenced AQP4 expression in the cerebral cortex and dorsal striatum.

Significance

Melatonin confers neuroprotection by protein kinase C mediated AQP4 inhibition in ischemic stroke.     

Melatonin effect on the ultrastructure of Ehrlich ascites tumor cells,lifetime and histopathology in Swiss mice

Aims

One of the models used for studying cancer is the Ehrlich ascites tumor (EAT) due to its ability to grow in liquid suspension, allowing a standard number of cells to be inoculated, growth quantification and regression of tumor mass. Among the oncostatic substances, melatonin has shown effectiveness in limiting the tumor cell proliferation. However, studies have shown contradictory effects of melatonin on the EAT. This study has investigated the melatonin effect on tumor growth, time and survival percentage, ultrastructure and metastasis of EAT cells in mice submitted or not to pinealectomy.

Main methods

Animals were inoculated with 5 × 10⁶ cells/mL and treated or not with exogenous melatonin with doses of at 150 and 300 μg/30 g animal weight for 12 days. Melatonin significantly reduced the abdominal circumference, volume of ascites liquid and EAT-cell viability, raising rates of time and mice survival percentage.

Key findings

Ultrastructurally, the melatonin treatment revealed changes in the shape of cells, the cell surface showed numerous projections, some bifurcated, cytoplasmic vacuolation, mitochondrial degeneration and nuclear fragmentation, peculiar characteristics of apoptosis. Histopathology revealed no metastasis in the liver, small intestine and large intestine in any of the animals in the experimental groups; however this process was evident in the lungs and kidneys, being inhibited by melatonin administration.

Significance

Thus, we can conclude that doses of 150 and 300 μg/30 g of melatonin for 12 consecutive days have a very effective oncostatic and cytotoxic activity on EAT cells in mice.     

Carbamoyl-PROXYL-enhanced MRI detects very small disruptions in brain vascular permeability induced by dietary cholesterol

Background

Gd-DTPA-enhanced magnetic resonance imaging (MRI) is a conventional method for non-invasive investigation of blood-brain-barrier (BBB) permeability in animal models. It allows the visualization of serious injury to the BBB. We developed a novel approach for detecting very small disruptions in BBB permeability induced by dietary cholesterol by using carbamoyl-PROXYL (CMP) as an MRI contrast probe.

Methods

Mice were separated into two groups: normal diet (ND-mice) and high cholesterol diet (CD-mice). MRI-signal dynamics, plasma cholesterol, matrix metalloproteinase (MMP-9, MMP-2), and the white blood cell profile were analyzed. For the MRI analysis, two regions-of-interest (ROI) were selected: brain (ROI-1) and surrounding area (ROI-2).

Results

In the ROI-2 of ND-mice, CMP- or Gd-enhanced MRI-signal followed typical kinetics with a half-life of signal decay (τ_1/2) ~ 8 or ~ 15 min, respectively. In CD-mice, the MRI-signal increased continuously without decay.In the ROI-1 of ND- and CD-mice, MRI-signal enhancement was not detected by Gd-DTPA. In the ROI-1 of ND-mice, CMP-induced MRI-signal enhancement was negligible, while in CD-mice, it was significant (τ_1/2 > 15 min).Hypercholesterolemia increased the plasma levels of MMP-9 and neutrophils.

Conclusions

Hypercholesterolemia increases vascular permeability, which is mediated by MMP-9 and neutrophils.

General significance

Even very small disruptions in brain vascular permeability could be detected by CMP-enhanced MRI but not by Gd-DTPA-enhanced MRI.     

Continuous oral administration of atorvastatin ameliorates brain damage after transient focal ischemia in rats

Aims

Pre-treatment with statins is known to ameliorate ischemic brain damage after experimental stroke, and is independent of cholesterol levels. We undertook pre- vs post-ischemic treatment with atorvastatin after focal cerebral ischemia in rats.

Main methods

Male Sprague–Dawley rats underwent transient 90-min middle cerebral artery occlusion (MCAO). Atorvastatin (20 mg/kg/day) or vehicle was administered orally. Rats were divided into vehicle-treated, atorvastatin pre-treatment, atorvastatin post-treatment, and atorvastatin continuous-treatment groups. In the pre-treatment, rats were given atorvastatin or vehicle for 7 days before MCAO. In the post-treatment, rats received atorvastatin or vehicle for 7 days after MCAO. Measurement of infarct volume, as well as neurological and immunohistochemical assessments, were done 24 h and 7 days after reperfusion.

Key findings

Each atorvastatin-treated group demonstrated significant reductions in infarct and edema volumes compared with the vehicle-treated group 24 h after reperfusion. Seven days after reperfusion, infarct volumes in the post-treatment group and continuous-treatment group (but not the pre-treatment group) were significantly smaller than in the vehicle-treated group. Only the continuous-treatment group had significantly improved neurological scores 7 days after reperfusion compared with the vehicle group. Post-treatment and continuous-treatment groups had significantly decreased lipid peroxidation, oxidative DNA damage, microglial activation, expression of tumor necrosis factor-alpha, and neuronal damage in the cortical ischemic boundary area after 7 days of reperfusion.

Significance

These results suggest that continuous oral administration (avoiding withdrawal) with statins after stroke may reduce the extent of post-ischemic brain damage and improve neurological outcome by inhibiting oxidative stress and inflammatory responses.     

Effect of repeated allogeneic bone marrow mononuclear cell transplantation on brain injury following transient focal cerebral ischemia in rats

Aims

Transplantation of bone marrow mononuclear cells (BMMCs) exerts neuroprotection against cerebral ischemia. We examined the therapeutic timepoint of allogeneic BMMC transplantation in a rat model of focal cerebral ischemia, and determined the effects of repeated transplantation outside the therapeutic window.

Main methods

Male Sprague–Dawley rats were subjected to 90 minute focal cerebral ischemia, followed by intravenous administration of 1 × 10⁷ allogeneic BMMCs or vehicle at 0, 3 or 6 h after reperfusion or 2 × 10⁷ BMMCs 6 h after reperfusion. Other rats administered 1 × 10⁷ BMMCs at 6 h after reperfusion received additional BMMC transplantation or vehicle 9 h after reperfusion. Infarct volumes, neurological deficit scores and immunohistochemistry were evaluated 24 or 72 h after reperfusion.

Key findings

Infarct volumes at 24 h were significantly decreased in transplantation rats at 0 and 3 h, but not at 6 h, after reperfusion, compared to vehicle-treatment. Even high dose BMMC transplantation at 6 h after reperfusion was ineffective. Repeated BMMC transplantation at 6 and 9 h after reperfusion reduced infarct volumes and significantly improved neurological deficit scores at 24 and 72 h. Immunohistochemistry showed repeated BMMC transplantation reduced ionized calcium-binding adapter molecule 1, 4-hydroxy-2-nonenal and 8-hydroxydeoxyguanosine expression at 24 and 72 h after reperfusion.

Significance

Intravenous allogeneic BMMCs were neuroprotective following transient focal cerebral ischemia, and the therapeutic time window of BMMC transplantation was > 3 h and < 6 h after reperfusion in this model. Repeated transplantation at 6 and 9 h after reperfusion suppressed inflammation and oxidative stress in ischemic brains, resulting in improved neuroprotection.     

Circulating hormone adrenomedullin and its binding protein protect neural cells from hypoxia-induced apoptosis

Background

Brain ischemia is the underlying cause of neuron death during stroke and brain trauma. Neural cells exposed to ischemia can undergo apoptosis. Adrenomedullin (AM) in combination with its enhancing binding protein, AMBP-1, has been shown to reduce tissue damage in inflammation.

Methods

To evaluate a beneficial effect of AM/AMBP-1 administration in brain ischemia, we employed an in vitro model of neuronal hypoxia using differentiated human neuroblastoma SH-SY5Y cells.

Results

After exposure to 1% O₂ for 20 h, neural cells were injured with decreased ATP levels and increased LDH release. Pre-administration of AM/AMBP-1 significantly reduced hypoxia-induced cell injury. Moreover, AM/AMBP-1 treatment reduced the number of TUNEL-positive cells and activation of caspase-3, compared to cells exposed to hypoxia alone. AM/AMBP-1 prevented a reduction of cAMP levels and protein kinase A (PKA) activity in neural cells after hypoxia exposure. Correspondingly, an elevation of cAMP levels by forskolin protected neural cells from hypoxia-induced injury. Inhibition of PKA by KT5720 abolished the protective effect of AM/AMBP-1 on hypoxia-induced apoptosis.

Conclusions

AM/AMBP-1 elevates cAMP levels, followed by activating PKA, to protect neural cells from the injury caused by hypoxia.

General significance

AM/AMBP-1 may be used as therapeutic agents to prevent neuron damage from brain ischemia.     

Anti-inflammatory effects of anethole in lipopolysaccharide-induced acute lung injury in mice

Aims

Anethole, the major component of the essential oil of star anise, has been reported to have antioxidant, antibacterial, antifungal, anti-inflammatory, and anesthetic properties. In this study, we investigated the anti-inflammatory effects of anethole in a mouse model of acute lung injury induced by lipopolysaccharide (LPS).

Main methods

BALB/C mice were intraperitoneally administered anethole (62.5, 125, 250, or 500 mg/kg) 1 h before intratracheal treatment with LPS (1.5 mg/kg) and sacrificed after 4 h. The anti-inflammatory effects of anethole were assessed by measuring total protein and cell levels and inflammatory mediator production and by histological evaluation and Western blot analysis.

Key findings

LPS significantly increased total protein levels; numbers of total cells, including macrophages and neutrophils; and the production of inflammatory mediators such as matrix metalloproteinase 9 (MMP-9), tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and nitric oxide (NO) in bronchoalveolar lavage fluid. Anethole (250 mg/kg) decreased total protein concentrations; numbers of inflammatory cells, including neutrophils and macrophages; and the inflammatory mediators MMP-9, TNF-α and NO. In addition, pretreatment with anethole decreased LPS-induced histopathological changes. The anti-inflammatory mechanism of anethole in LPS-induced acute lung injury was assessed by investigating the effects of anethole on NF-κB activation. Anethole suppressed the activation of NF-κB by blocking IκB-α degradation.

Significance

These results, showing that anethole prevents LPS-induced acute lung inflammation in mice, suggest that anethole may be therapeutically effective in inflammatory conditions in humans.     

Update meta-analysis on MMP-7 − 181A>G polymorphism and cancer risk: Evidence from 25 studies

Background

The matrix metalloproteinase (MMP) can degrade various components of the extracellular matrix and its functional genetic polymorphism may be associated with cancer development. The common MMP-7 (− 181A>G) genetic polymorphism has been reported to be functional and may contribute to genetic susceptibility to cancers. However, the association between MMP-7 (− 181A>G) and cancer risk remains inconclusive.

Methods

To better understand the role of MMP-7 (− 181A>G) polymorphism in global cancer, we conducted this comprehensive meta-analysis encompassing 6392 cases and 7665 controls.

Results

Overall, the MMP-7 (− 181A>G) polymorphism was associated with higher cancer risk. In the stratified analyses, significant associations were found between the MMP-7 (− 181A>G) polymorphism and gastric cancer, ESCC and gynecologic cancer. We also observed that the GG genotype might modulate colorectal cancer risk comparing with the AA genotype (OR = 1.31[1.02–1.69]). Moreover, a significantly increased cancer risk was found among Asian populations. When stratified by study design, significantly elevated susceptibility to cancer was found among population-based studies.

Conclusions

These findings suggested that the MMP-7 (− 181A>G) genetic polymorphism may contribute to the susceptibility of cancers, especially among Asian population.     

Penetration of verapamil across blood brain barrier following cerebral ischemia depending on both paracellular pathway and P-glycoprotein transportation

Background

Blood brain barrier (BBB) dysfunction is a common facet of cerebral ischemia, and the alteration of drug transporter, P-glycoprotein (P-gp), has been documented.

Aims

This study explores influence of damaged BBB and elevated P-gp on cerebral verapamil penetration after ischemia both in vivo and in vitro.

Methods

Middle cerebral artery occlusion (MCAO) induced ischemia/reperfusion (I/R) of rats, and Na₂S₂O₄ induced hypoxia/reoxygenation (H/R) damage of rat brain mirovessel endothelial cells (RBMECs) respectively, served as BBB breakdown model in vivo and in vitro. Evans-Blue (EB) extravagation and ¹²⁵I-albumin were used to quantify BBB dysfunction; UPLC–MS/MS analytical method was performed to determine accurately the concentration of verapamil in brain tissue and cell. Flow cytometry, immunohistochemistry and western blotting were applied to evaluate transport function and protein expression of P-gp.

Results

Overexpressed ICAM-1 and MMP-9 mediated BBB dysfunction after ischemia, which induced EB leakage and ¹²⁵I-albumin uptake increase. Enhanced accumulation of verapamil in brain tissue, but intracellular concentration reduced evidently after H/R injury. Transcellular transportation of verapamil elevated when P-gp function or expression was inhibited after H/R injury.

Conclusion

These data indicated that BBB penetration of verapamil under ischemia condition was not only depending on BBB breakdown, but also regulated by P-gp.     

Characterization of ecto-ATPase activity in the surface of LLC-PK1 cells and its modulation by ischemic conditions

Background

The concentration of extracellular nucleotides is regulated by enzymes that have their catalytic site facing the extracellular space, the so-called ecto-enzymes.

Methods

We used LLC-PK1 cells, a well-characterized porcine renal proximal tubule cell line, to biochemically characterize ecto-ATPase activity in the luminal surface. The [γ-³²P]Pi released after reaction was measured in aliquots of the supernatant by liquid scintillation.

Results

This activity was linear with time up to 20 min of reaction and stimulated by divalent metals. The ecto-ATPase activity measured in the presence of 5 mM MgCl₂ was (1) optimum at pH 8, (2) insensitive to different inhibitors of intracellular ATPases, (3) inhibited by 1 mM suramin, an inhibitor of ecto-ATPases, (4) sensitive to high concentrations of sodium azide (NaN₃) and (5) also able to hydrolyze ADP in the extracellular medium. The ATP:ADP hydrolysis ratio calculated was 4:1. The ecto-ADPase activity was also inhibited by suramin and NaN₃. The dose–response of ATP revealed a hyperbolic profile with maximal velocity of 25.2 ± 1.2 nmol Pi x mg^− 1 x min^− 1 and K_0.5 of 0.07 ± 0.01 mM. When cells were submitted to ischemia, the E-NTPDase activity was reduced with time, achieving 71% inhibition at 60 min of ischemia.

Conclusion

Our results suggest that the ecto-ATPase activity of LLC-PK1 cells has the characteristics of a type 3 E-NTPDase which is inhibited by ischemia.

General Significance

This could represent an important pathophysiologic mechanism that explains the increase in ATP concentration in the extracellular milieu in the proximal tubule during ischemia.     

Oxidation of melatonin by AAPH-derived peroxyl radicals: Evidence of a pro-oxidant effect of melatonin

Background

Melatonin is well-established as a powerful reducing agent of oxidant generated in the cell medium. We aimed to investigate how readily melatonin is oxidized by peroxyl radicals ROO⋅ generated by the thermolysis of 2,2′-azobis(2-amidinopropane) hydrochloride (AAPH) and the role of glutathione (GSH) during the reaction course.

Methods

Chromatographic, mass spectroscopy, and UV–visible spectrometric techniques were used to study the oxidation of melatonin by ROO⋅ or horseradish peroxidase (HRP)/H₂O₂. Our focus was the characterization of products and the study of features of the reaction.

Results

We found that N¹-acetyl-N²-formyl-5-methoxykynuramine (AFMK) and a monohydroxylated derivative of melatonin were the main products of the reaction between melatonin and ROO⋅. Higher pH or saturation of the medium with molecular oxygen increased the yield of AFMK but did not affect the reaction rate. Melatonin increased the depletion of intracellular GSH mediated by AAPH. Using the HRP/H₂O₂ as the oxidant system, the addition of melatonin promoted the oxidation of GSH to GSSG.

Conclusions

These results show, for the first time, that melatonin radical is able to oxidize GSH.

General significance

We propose that this new property of melatonin could explain or be related to the recently reported pro-oxidant activities of melatonin.     

MMP-1 promoter genotype and haplotype association with posterior tibial tendinopathy

Purpose

Posterior tibial tendon (PTT) is particularly vulnerable and its insufficiency is recognized as the main cause of adult acquired flatfoot. Some patients have a predisposition without clinically recognized cause, suggesting that individual characteristics play an important role in tendinopathy. The objective of the present study is to investigate the association of − 519 (rs1144393) matrix metalloproteinase-1 (MMP-1) polymorphism and the − 1607 (rs1799750) and − 519 MMP-1 haplotypes and risk of PTT dysfunction.

Methods

The test group included 50 females who presented PTT dysfunction Grade 2 or 3, and who were submitted to surgical treatment, with histopathological examination of the tendon and magnetic resonance image (MRI) confirming tendinopathy, while the control group was 100 asymptomatic women who present intact PTT at MRI. We analyzed functional polymorphisms MMP-1 and their haplotypes using polymerase chain reaction and restriction fragment length analysis.

Results

There was a significant difference in the presence of the different alleles and genotypes between the control group and test group for the MMP-1 gene (p ≤ 0.01). The G allele of the − 519 MMP-1 polymorphism increased susceptibility to degeneration in the PTT tendon and seems to be a genetic risk factor. Global haplotype analysis indicated a significant difference between both groups (p < 0.0001). Haplotypes G–2G and A–2G had statistically significant risk effect on PTT insufficiency. G–2G, p < 0.001; OR = 5.72 (CI, 2.84–11.52) and A–2G p = 0.002, OR = 3.95 (CI, 1.65–9.44).

Conclusion

According to our results, − 519 MMP-1 isolated and − 1607/− 519 MMP-1 haplotypes are associated to tendinopathy in posterior tibial tendon.     

Effects of intravenous cariporide on release of norepinephrine and myoglobin during myocardial ischemia/reperfusion in rabbits

Aims

To examine the effects of cariporide, a Na⁺/H⁺ exchanger-1 inhibitor, on cardiac norepinephrine (NE) and myoglobin release during myocardial ischemia/reperfusion by applying a microdialysis technique to the rabbit heart.

Main methods

In anesthetized rabbits, two dialysis probes were implanted into the left ventricular myocardium and were perfused with Ringer's solution. Cariporide (0.3 mg/kg) was injected intravenously, followed by occlusion of the left circumflex coronary artery. During 30-min coronary occlusion followed by 30-min reperfusion, four consecutive 15-min dialysate samples (two during ischemia and two during reperfusion) were collected in vehicle and cariporide-treated groups. Dialysate myoglobin and NE concentrations were measured by immunochemistry and high-performance liquid chromatography, respectively.

Key findings

Dialysate myoglobin and NE concentrations increased significantly during myocardial ischemia/reperfusion in both vehicle and cariporide-treated groups (P < 0.01 vs. baseline). In cariporide-treated group, dialysate myoglobin concentrations were significantly lower than those in vehicle group throughout ischemia/reperfusion (P < 0.01 at 0–15 min of ischemia, P < 0.05 at 15–30 min of ischemia, P < 0.01 at 0–15 min of reperfusion, and P < 0.01 at 15–30 min of reperfusion). However, dialysate NE concentrations in cariporide-treated group were lower than those in vehicle group only during ischemia (P < 0.01 at 0–15 min of ischemia, and P < 0.05 at 15–30 min of ischemia).

Significance

When administered before ischemia, cariporide reduces myoglobin release during ischemia/reperfusion and decreases NE release during ischemia.     

Up-regulation of adenylylcyclases I and II induced by long-term adaptation of rats to morphine fades away 20 days after morphine withdrawal

Background

Activation of adenylyl cyclase (AC) by prolonged exposure of mammalian organism to morphine was demonstrated in previous studies of mechanism of action of this drug. However, expression level of individual AC isoforms was not analyzed in crucial cell structure, plasma membrane (PM).

Methods

Rats were adapted to morphine for 10 days and sacrificed 24 h (group + M10) or 20 days (+ M10/−M20) after the last dose. Control animals were sacrificed in parallel with morphine-treated (groups − M10 and (− M10/−M20)). Percoll®-purified PM were isolated from brain cortex and analyzed by immunoblotting and specific radioligand binding.

Results

ACI (ACII) was increased 8× (2.5×) in morphine-adapted rats (+ M10) when compared with controls (− M10). Increase of ACI and II by long-term adaptation to increasing doses of morphine represented a specific effect as the amount of ACIII–ACIX, of prototypical PM marker, Na, K-ATPase and of trimeric G protein α and β subunits was unchanged. Increase of ACI and II was not detected in PM isolated from group (+ M10/−M20). Thus, the marked increase of ACI and ACII faded away 20 days since the last dose of morphine.

Conclusions

We assume that the specific increase in expression level of ACI and ACII in brain cortex of morphine-adapted rats proceeds as a compensatory, homeostatic response to prolonged exposure to inhibitory drug, morphine.

General significance

Our findings demonstrate that the dramatic and specific change of the crucial component of the opioid receptor cascade in brain cortex, manifested as an increase in PM level of ACI and II, is reversible.     

PAC-1 and isatin derivatives are weak matrix metalloproteinase inhibitors

Background

Dysregulation of apoptotic cell death is observed in a large number of pathological conditions. As caspases are central enzymes in the regulation of apoptosis, a large number of procaspase-activating compounds (PAC-1 derivatives) and inhibitors (isatin derivatives) have been developed. Matrix metalloproteinases (MMPs) have been shown to have a dual role in apoptosis. Hence compounds that either activate or inhibit caspases should ideally not affect MMPs. As many PAC-1 derivatives contain a zinc chelating ortho-hydroxy N-acyl hydrazone moiety and isatin derivatives has two carbonyl groups on the indole core, it was of interest to determine to which extent these compounds can inhibit MMPs.

Methods

Eight PAC-1 and five isatin derivatives were docked into MMP-9 and MMP-14. The same compounds were synthesized, characterized, purified and tested as inhibitors of MMP-9 and MMP-14, using fluorescence quenched peptide and biological substrates. Some of the compounds were also tested for fluorescence quenching.

Results

Molecular docking suggested that the different compounds can bind to the MMP active sites. However, kinetic studies showed that neither of these compounds was a strong MMP inhibitor. IC₅₀ values over 100 μM were obtained after the enzyme activities were corrected for quenching. These IC₅₀ values are far above the concentrations needed to activate or inhibit the caspases.

Conclusion

The use of PAC-1 and isatin derivatives against caspases should have little or no effect on the activity of MMPs.

General significance

Activators and inhibitors of caspases are important potential therapeutic agents for several diseases such as cancer, diabetes and neurodegenerative disorders.     

Quercetin glucosides promote ischemia-induced angiogenesis,but do not promote tumor growth

Aims

Dietary flavonoid intake shows a significant inverse association with mortality from coronary heart disease, incidence of myocardial infarction and stroke. Quercetin is one of the most common flavonoids in our diet and has several favorable biological activities. Quercetin glucosides, which are enzymatically trans-glycosylated isoquercitrin, have high water-solubility and bioavailability compared with quercetin. Here, we investigated the effects of quercetin glucosides on collateral development in a murine hindlimb ischemia model.

Main methods

We induced hindlimb ischemia in 24- to 32-week-old male C3H/HeJ mice by resecting the right femoral artery. Then, 0.5% carboxymethyl cellulose (control) or quercetin glucosides (100 mg/kg/day) were administered daily by gavage. Blood flow was monitored weekly by laser Doppler imaging.

Key findings

Recovery of blood flow to the ischemic leg was significantly enhanced by quercetin glucosides (blood flow ratio at 4 weeks: control, 0.57 ± 0.11; quercetin glucosides, 0.95 ± 0.10, p < 0.05). Furthermore, anti-CD31 immunostaining revealed that quercetin glucosides increased capillary density in the ischemic muscle (control, 200 ± 24/mm²; quercetin glucosides, 364 ± 41/mm², p < 0.01). Quercetin glucosides did not promote tumor growth. The beneficial effect of quercetin glucosides was abrogated in eNOS-deficient mice.

Significance

These results suggest that quercetin glucosides may have therapeutic potential to promote angiogenesis in ischemic tissue.     

Macrophage elastase (MMP-12) in expanding murine adipose tissue

Background

Matrix metalloproteinases (MMPs) are known to play a role in adipose tissue development, but little information is available on the role of individual proteinases. Expansion of adipose tissue is associated with an increased macrophage content. Macrophage elastase (MMP-12) has an important role in macrophage infiltration, which induces pro-inflammatory effects in adipose tissue.

Methods

The role of MMP-12 was investigated in adipose tissues of MMP-12 deficient and wild-type control mice kept on normal chow or on high fat diet for 15 weeks.

Results

MMP-12 deficiency had no significant effect on total body weight or on subcutaneous (SC) or gonadal (GON) adipose tissue mass. Adipocyte and blood vessel size and density in SC and GON adipose tissues of obese mice were also comparable in MMP-12 deficient and control mice. Macrophage infiltration in SC and GON adipose tissues was not affected by MMP-12 deficiency, but the amount of crown-like structures (CLS) was significantly lower. MMP-12 deficiency did not affect elastin content in the extracellular matrix of SC or GON adipose tissue.

Conclusions

Adipose tissue mass and composition in mice with nutritionally induced obesity was not markedly affected by MMP-12 deficiency, except for an apparently lower degree of CLS.

General Significance

MMP-12 does not seem to be essential for macrophage infiltration in adipose tissue, but contributes to the formation of CLS surrounding moribund adipocytes.     

Cholecalciferol (vitamin D 3) improves cognitive dysfunction and reduces inflammation in a rat fatty liver model of metabolic syndrome

Aim

The aim of this study was to examine the effects of cholecalciferol on systemic inflammation and memory in the setting of fatty liver disease in rats.

Materials and methods

To induce the development of fatty liver disease, the rats were fed a 35% fructose solution over 8 weeks. Group I (n = 6) was designated as the control group and fed with standard rat chow. Group II (n = 6) was provided with, standard rat chow, and 0.3 μg/kg/day of oral cholecalciferol over a duration of 2 weeks. In addition to standard rat chow, group III (n = 6) and group IV (n = 6) were given 4 mL of the 35% fructose solution per day via oral gavage for 8 weeks. However, group IV was also given 0.3 μg/kg/day of oral cholecalciferol over 2 weeks. After the treatment period, passive avoidance tasks were performed by all groups. The liver and brain were harvested for subsequent biochemical and histopathologic analyses.

Key findings

The development of fatty liver extends the memory latency period of passively avoiding tasks after 1 trial. Moreover, there were increases in brain TNF-α and plasma MDA levels according to two-way analysis of variance. Cholecalciferol supplementation decreased the latency period of passively avoiding tasks in rats with hepatosteatosis, and also significantly reduced brain TNF-α and plasma MDA levels.

Significance

Fatty liver may contribute to the development of systemic inflammation, which affects cognition and causes deficits in memory; however, the anti-inflammatory and antioxidant properties of vitamin D may improve the cognitive function of rats with hepatosteatosis.