Osseointegration of hydroxyapatite and remodeling‐resorption of tricalciumphosphate ceramics

Background: Cancellous bone defects surrounded by still intact bone structures never heal. Ceramics offer a solution providing osteoconductive scaffolds. Purpose: The purpose of the study is to evaluate whether structured β‐TCP and HA implants can reconstruct cancellous bone defects, which role micro‐ and macro‐porosity, stiffness and surface area play; finally the indication for both materials based on its resorbability. Material & Methods: 10 German Shepard dogs were operated on both tibial heads implanting shell‐like fully interconnected ceramic cylinders, using a wet grinding hollow drill coated with diamonds. β‐TCP was compared with HA. A polychromatic sequential labelling with 4 different fluorochromes controlled bone formation dynamics. Non‐decalcifying histology after perfusion fixation and vessel casting was performed. μ‐CT was combined with high resolution microradiography and histology on thin ground crossections. The stages after 6 weeks, 2, 3, 4 months and 15 months were evaluated. Results: In spite of osseointegration of HA and β‐TCP, the osseointegration of both materials was completely different. Both shell‐like bone void fillers were osseointegrated in a sandwich‐like manner. HA yielded primarily a reinforcement of the recipient's cancellous‐bone bed and full osseointegration after 4 months, whereas β‐TCP‐implants were fully osseointegrated after 6 weeks. HA did not show signs of resorption. The resorption of the β‐TCP resulted during remodelling. The final stage showed restitution “ad integrum” of the β‐TCP defects with a physiological architecture, whereas HA was integrated in the cancellous bone construction providing 600 μm measuring macropores showing osteoinductive properties. Microsc. Res. Tech. 76:370–380, 2013. © 2013 Wiley Periodicals, Inc.     

Two‐photon laser scanning microscopy as a useful tool for imaging and evaluating macrophage‐, IL‐4 activated macrophage‐ and osteoclast‐based In Vitro degradation of beta‐tricalcium phosphate bone substitute material

Two‐photon microscopy is an innovative technology that has high potential to combine the examination of soft and hard tissues in vitro and in vivo. Calcium phosphates are widely used substitutes for bone tissue engineering, since they are degradable and consequently replaced by newly formed tissue. It is well known that osteoclasts are responsible for the resorption processes during bone remodelling. We hypothesize that also macrophages are actively involved in the resorption process of calcium phosphate scaffolds and addressed this question in in vitro culture systems by two‐photon laser scanning microscopy. Beta‐tricalcium phosphate specimens were incubated with (1) macrophages, (2) interleukin‐4 activated macrophages, and (3) osteoclasts for up to 21 days. Interestingly, macrophages degraded beta‐tricalcium phosphate specimens in an equivalent fashion compared to osteoclasts and significantly more than IL‐4 activated macrophages. An average of ～32% of the macrophages was partially filled with ceramic material while this was 18% for osteoclasts and 9% for IL‐4 activated macrophages. For the first time by applying two‐photon microscopy, our studies show the previously unrecognized potential of macrophages to phagocytose ceramic material, which is expected to have implication on osteoconductive scaffold design. Microsc. Res. Tech. 77:143–152, 2014. © 2013 Wiley Periodicals, Inc.     

Effects of TGF‐β1 on mineralization mediated by rat calvaria‐derived osteogenic cells

In this study, we have analyzed the viability and cell growth, as well as, the mineralization of extracellular matrix (ECM) by alizarin red and von Kossa staining of calvaria‐derived osteogenic cultures, treated with TGF‐β1 alone or associated with Dex comparing with acid ascorbic (AA) + β‐glicerophosphate (βGP) (positive mineralization control). The expression of the noncollagenous proteins bone sialoprotein (BSP), osteopontin (OPN) and fibronectin (FN) were evaluated by indirect immunofluorescence. In addition, the main ultrastructural morphological findings were assessed by transmission electron microscopy. Osteogenic cells were isolated of calvaria bone from newborn (2‐day‐old) Wistar rats were treated with TGF‐β1 alone or with dexamethasone for 7, 10, and 14 days. As positive mineralization control, the cells were supplemented only with AA+ βGP. As negative control, the cells were cultured with basal medium (α‐MEM + 10%FBS + 1%gentamicin). The treatment with TGF‐β1, even when combined with Dex, decreased the viability and cell growth when compared with the positive control. Osteoblastic cell cultures were positive to alizarin red and von Kossa stainings after AA + βGP and Dex alone treatments. Positive immunoreaction was found for BSP, OPN and FN in all studied treatments. Otherwise, when the cell cultures were supplemented with TGF‐β1 and TGF‐β1 + Dex, no mineralization was observed in any of the studied periods. These present findings suggest that TGF‐β1, in the studied in vitro doses, inhibits the proliferation and differentiation of osteoblastic cells by impairment of nodule formation.     

Role of spermatogonial stem cells extract in transdifferentiation of 5‐Aza‐2′‐deoxycytidine‐treated bone marrow mesenchymal stem cells into germ‐like cells

As one of the induced pluripotent stem cells (iPSCs) methods, spermatogonial stem cells (SSC_S) extract is considered as new approach in stem cell therapy of infertility. 5‐aza‐2′‐deoxycytidine (5‐aza‐dC) inhibits methyltransferase enzyme, and induces gene reprogramming; herein, the effects of SSC_S extract incubation in 5‐aza‐dC‐treated bone marrow mesenchymal stem cells (BMMSCs) has been surveyed. BMMSCs were isolated from femurs of three to four weeks old male NMRI mice, and the cells at passage three were treated with 2 µM 5‐aza‐dC for 72 hours. SSCs were isolated, cultured, and harvested at passage three to collect SSC_S extract; BMMSCs were then incubated with SSC_S extract in the three time periods: 72 hours, one week and two weeks. There were five groups: control, sham, extract, 5‐aza‐dC and extract‐5‐aza‐dC. After one week of incubation, flow cytometry and real‐time polymerase chain reaction (PCR) exhibited high levels of expression for β1‐ and α6‐integrins and promyelocytic leukaemia zinc finger (PLZF) in extract and extract‐5‐aza‐dC groups (P < 0.05 vs. control and 5‐aza‐dC), and cells in these two groups had two forms of morphology, round and fusiform, similar to germ‐like cells. 5‐aza‐dC had no significant effects during the three time periods of evaluation. These data disclose the effectiveness of SSCs extract incubation in transdifferentiation of BMMSCs into germ‐like cells; this strategy could introduce a new approach for treatment of male infertility in clinic. Microsc. Res. Tech. 79:365–373, 2016. © 2016 Wiley Periodicals, Inc.     

Quantitative and reliable in vitro method combining scanning electron microscopy and image analysis for the screening of osteotropic modulators

The increased generation and up-regulated activity of bone resorbing cells (osteoclasts) play a part in the impairment of bone remodeling in many bone diseases. Numerous drugs (bisphosphonates, calcitonin, selective estrogen receptor modulators) have been proposed to inhibit this increased osteoclastic activity. In this report, we describe a pit resorption assay quantified by scanning electron microscopy coupled with image analysis. Total rabbit bone cells with large numbers of osteoclasts were cultured on dentin slices. The whole surface of the dentin slice was scanned and both the number of resorption pits and the total resorbed surface area were measured. Resorption pits appeared at 48 h and increased gradually up to 96 h. Despite the observation of a strong correlation between the total resorption area and the number of pits, we suggest that area measurement is the most relevant marker for osteoclastic activity. Osteotropic factors stimulating or inhibiting osteoclastic activity were used to test the variations in resorption activity as measured with our method. This reproducible and sensitive quantitative method is a valuable tool for screening for osteoclastic inhibitors and, more generally, for investigating bone modulators.     

Short‐Term Response of Human Osteoblast‐Like Cells on Titanium Surfaces With Micro‐ and Nano‐Sized Features

Since the way that human bone cells behave on contact with different surfaces topographies seems to be crucial to osseointegration, the aim of the present study is to evaluate the participation of some micro‐ and nanosized features of Ti surfaces in the short‐term response of primary human osteoblast‐like cells (HOC). Surfaces were prepared as ground (G‐Ti), hydrofluoric acid etched (HF‐Ti), and sandblasted/HF‐etched (SLA‐Ti), and analyzed using both three‐dimensional (3D) profilometer and atomic force microscope (AFM). Cell morphology was assessed using scanning electron microscopy (SEM) after 4 and 24 h in culture. Cell viability, adhesion, and spreading were also evaluated 4 and 24 h after seeding over each surface. Data were compared by analysis of variance (ANOVA) complemented by Duncan test. Cell morphology, cell counting, and membrane integrity (Neutral Red, NR) were not affected by surface treatment at any time. However, HF‐Ti presented the smallest surface area and did not increase tetrazolium hydroxide (XTT) reduction from 4 to 24 h. On the other hand, a higher level of spreading was only found on the rougher and isotropic SLA‐Ti at 4 h. In conclusion, although all evaluated Ti surfaces allowed HOC short‐term adhesion, the finer topography introduced by HF as single treatment did not favor HOC mitochondrial activity and spreading. The rougher and more complex SLA surface seems to provide a better substrate for HOC short‐term response. SCANNING 34: 378‐386, 2012. © 2012 Wiley Periodicals, Inc.     

Investigation of mechanical and tribological properties of tribo‐layer of Ni3Al matrix composites

The sliding wear of Ni₃Al matrix composites with addition of 1.5 wt.% graphene nanoplates was studied through pin‐on‐disc wear testing. The spontaneous formation of a tribo‐layer produced during sliding wear was found to result in a deviation from Archard scaling and an unexpected high wear resistance that was not based on hardness alone. The tribo‐layer exhibited specific microstructural evolution with significant severe deformation and grain refinement after wear. In the grain refinement area, the accumulation of dislocations and an increase in misorientations were found to lead to strain hardening. For the plastic deformed area, reduction in the dislocation density inside the elongated ultrafine grains reduced strain hardening compared with the grain refinement area. It can be concluded that the deviation from Archard scaling occurred primarily as a result of the microstructural evolution of the tribo‐layer, resulting in the specific performance of mechanical and tribological properties of Ni₃Al matrix composites under cyclic sliding wear process. Copyright © 2016 John Wiley & Sons, Ltd.     

Technical note: Extreme‐pressure activity assessment of certain nitrogen and sulphur compounds in lithium‐based grease

In this paper we report on the preparation and extreme‐pressure (EP) activity assessment of certain substituted 1‐amino‐3‐aryl‐2,3‐dihydro‐2‐thioxo‐4,6‐(1H,5H)‐pyrimidinediones as additives in a lithium‐based grease. These additives significantly decreased the wear and friction and possessed the ability to increase load‐carrying capacities and weld loads. Two greases were prepared with the above additives, and both greases exhibited lower values of wear‐scar diameter at higher loads and higher values of weld load in the four‐ball test than the lithium‐based grease alone. The prepared greases also passed rust and corrosion, and oxidation tests.     

Preserving elemental content in adherent mammalian cells for analysis by synchrotron‐based x‐ray fluorescence microscopy

Trace metals play important roles in biological function, and x‐ray fluorescence microscopy (XFM) provides a way to quantitatively image their distribution within cells. The faithfulness of these measurements is dependent on proper sample preparation. Using mouse embryonic fibroblast NIH/3T3 cells as an example, we compare various approaches to the preparation of adherent mammalian cells for XFM imaging under ambient temperature. Direct side‐by‐side comparison shows that plunge‐freezing‐based cryoimmobilization provides more faithful preservation than conventional chemical fixation for most biologically important elements including P, S, Cl, K, Fe, Cu, Zn and possibly Ca in adherent mammalian cells. Although cells rinsed with fresh media had a great deal of extracellular background signal for Cl and Ca, this approach maintained cells at the best possible physiological status before rapid freezing and it does not interfere with XFM analysis of other elements. If chemical fixation has to be chosen, the combination of 3% paraformaldehyde and 1.5 % glutaraldehyde preserves S, Fe, Cu and Zn better than either fixative alone. When chemically fixed cells were subjected to a variety of dehydration processes, air drying was proved to be more suitable than other drying methods such as graded ethanol dehydration and freeze drying. This first detailed comparison for x‐ray fluorescence microscopy shows how detailed quantitative conclusions can be affected by the choice of cell preparation method.     

The osteogenic effects of swimming on bone mass,strength, and microarchitecture in rats with unloading‐induced bone loss

The effect of nonweight‐bearing exercise on osteoporotic bones remains controversial and inconclusive. The purpose of this study was to evaluate the effects of swimming on osteoporotic tibias of rats submitted to hindlimb suspension. Initially, 20 Wistar rats were used to confirm a significant bone loss following 21 days of unloading. Thirty rats were then divided into 3 groups and followed during 51 days: CON (nonsuspended rats), S + WB (suspended rats for 21 days and then released for regular weight‐bearing) and, S + Swim (suspended rats for 21 days and then released from suspension and submitted to swimming exercise). We observed that swimming exercise was effective at fully recovering the bone deterioration caused by suspension, with significant increments in BMD, bone strength and bone volume. On the other hand, regular weight‐bearing failed at fully restoring the bone loss induced by unloading. These results indicate that swimming exercise may be a potential tool to improve bone density, strength, and trabecular volume in tibias with bone loss induced by mechanical unloading in suspended rats. We conclude that this modality of activity could be beneficial in improving bone mass, strength, and architecture in osteoporotic individuals induced by disuse, such as bed rest or those exposed to microgravity, who may not be able to perform weight‐bearing exercises. Microsc. Res. Tech. 78:784–791, 2015. © 2015 Wiley Periodicals, Inc.     

Efficacy of ethylene‐diamine‐tetra‐acetic acid associated with chlorhexidine on intracanal medication removal: A scanning electron microscopy study

The aim of this study was to evaluate the effectiveness of 17% ethylene‐diamine‐tetra‐acetic acid (EDTA) used alone or associated with 2% chlorhexidine gel (CHX) on intracanal medications (ICM) removal. Sixty single‐rooted human teeth with fully formed apex were selected. The cervical and middle thirds of each canal were prepared with Gates Glidden drills and rotary files. The apical third was shaped with hand files. The specimens were randomly divided into two groups depending on the ICM used after instrumentation: calcium hydroxide Ca(OH)₂+CHX or Ca(OH)₂+sterile saline (SS). After seven days, each group was divided into subgroups according to the protocol used for ICM removal: instrumentation and irrigation either with EDTA, CHX+EDTA, or SS (control groups). All specimens were sectioned and processed for observation of the apical thirds by using scanning electron microscopy. Two calibrated evaluators attributed scores to each specimen. The differences between the protocols for ICM removal were analyzed with Kruskal‐Wallis and Mann‐Whitney U tests. Friedman and Wilcoxon signed rank tests were used for comparison between the score of debris obtained in each root canal third. Remains of Ca(OH)₂ were found in all specimens independently of the protocol and ICM used (P > 0.05). Seventeen percent EDTA showed the best results in removing ICM when used alone (P < 0.05), particularly in those associated with CHX. It was concluded that the chelating agent 17% EDTA significantly improved the removal of ICM when used alone. Furthermore, the type of the vehicle associated with Ca(OH)₂ also plays a role in the ICM removal. Microsc. Res. Tech. 77:735–739, 2014. © 2014 Wiley Periodicals, Inc.     

17‐beta‐estradiol analog inhibits cell proliferation by induction of apoptosis in breast cell lines

Microtubules are important targets when studying potential anticancer agents since disturbance of these microtubule dynamics results in cell cycle arrest and cell death. 2‐Methoxyestradiol is a naturally occurring metabolite that exerts antiproliferative activity and induces apoptosis. Due to limited biological accessibly and rapid metabolic degradation, several analogs were synthesized. This study investigated the antiproliferative influence of an 2‐methoxyestradiol analog, (8R, 13S, 14S, 17S)‐2‐Ethyl‐13‐methyl‐7, 8, 9, 11, 12,13, 14, 15, 16, 17‐decahydro‐6H‐cyclopenta[a]phenanthrane‐3, 17‐diyl bis(sulfamate) (EMBS) on cell proliferation, morphology and apoptosis induction in a estrogen receptor‐positive breast adenocarcinoma cells line (MCF‐7), estrogen receptor‐negative highly metastatic breast cell line (MDA‐MB‐231) and a non‐tumorigenic breast epithelial cell line (MCF‐12A). Spectrophotometry results indicated that EMBS exerted differential antiproliferative activity in the three cell lines. Cell growth of the breast adenocarcinoma and highly metastatic breast cell line reached a plateau effect at 0.4 μM after 24 h of exposure. Light microscopy and polarization‐optical transmitted light differential interference contrast demonstrated compromised cell density, cells blocked in metaphase and the presence of apoptotic characteristics after EMBS exposure for 24 h in all three cell lines. Transmission electron microscopy and scanning electron microscopy revealed hallmarks of apoptosis namely the presence of apoptotic bodies, shrunken cells and cell debris in EMBS‐exposed cells. This investigation demonstrated that EMBS does exert antimitotic activity and induces apoptosis contributing to elucidating the signal transduction of EMBS in tumorigenic and non‐tumorigenic breast cell lines. Findings warrant in‐depth analysis of specific targets in vitro and subsequent in vivo investigation for anticancer therapy. Microsc. Res. Tech. 77:236–242, 2014. © 2014 Wiley Periodicals, Inc.     

Methodological assessment of acid‐etching for visualizing the osteocyte lacunar‐canalicular networks using scanning electron microscopy

Osteocytes are the most abundant of the bone cells. Each osteocyte is contained within its own lacuna and connected to adjacent osteocytes via fillipodial processes, which form an intricate network of canaliculi within the matrix. Studying this intricate network of cells and their processes is difficult, because it exists embedded within a densely mineralized matrix. Scanning electron microscopy (SEM) has been shown to be a useful tool for visualizing this cellular network, yet the techniques involved for preparing specimens has not been systematically explored. The goal of this study was to investigate how variations in acid‐etching, both etching media and etching duration, affect SEM‐based visualization of the osteocyte lacunar–canalicular network. Bone samples were embedded in plastic and then acid etched in either 9% (10, 20, 40, and 60 s durations) or 37% (5, 10, and 15 s) phosphoric acid. Specimens were imaged using SEM, and qualitative evaluation of the lacunar–canalicular network was undertaken. Our findings show acid etchingwith a 9% phosphoric acid solution for 20 s provided the most favorable visualization of the osteocyte lacunar–canalicular network. Microsc. Res. Tech., 2010. © 2009 Wiley‐Liss, Inc.     

Involution dependent changes in distribution and localization of bax,survivin, caspase‐3, and calpain‐1 in the rat endometrium

The endometrial layer of the uterus is characterized by continuous cycle of cell growth and apoptosis in response to hormonal changes. Apoptosis is regulated by several apoptotic regulators, but their significance in involuting uterus has not been well understood. For that reason, aim of this study was to investigate possible role of apoptosis‐related proteins (bax and survivin) and enzymes (caspase‐3 and calpain‐1) in the involuting uterus of the rat, using immunohistochemistry. Our results indicated cytoplasmic and nuclear immunostaining for bax, caspase‐3, calpain‐1 and survivin proteins were found in the endometrial epithelium and stromal cells such as fibroblasts, mast cells and macrophages, and blood vessels; however, calpain‐1 immunoreactivity in the endometrial fibroblast was quite weak or absent. Supranuclear punctate bax immunolabelling was also observed in the endometrial fibroblasts and luminal and glandular epithelial cells from days 1st and 3rd following parturition, respectively. Although survivin was localized in the apical cytoplasm underneath the apical membrane of the luminal epithelium on the 1st and 3rd days, it was also localized in the apicolateral membrane and basal cytoplasm on the 10th and 15th days of involution. Immunostainigs demonstrated that expression patterns of all examined proteins varied with structural changes in the luminal epithelium, and number of immunopositive fibroblasts for bax, caspase‐3 and survivin increased with advance of postpartum days and reached a maximum on postpartum days 10 and 15. These results suggest that the process of postpartum involution of endometrium may be regulated by apoptotic and non‐apoptotic activity of bax, caspase‐3, calpain‐1, and survivin. Microsc. Res. Tech. 79:285–297, 2016. © 2016 Wiley Periodicals, Inc.     

Region‐specific localization of NOS isoforms and NADPH‐diaphorase activity in the intratesticular and excurrent duct systems of adult domestic cats (Felis catus)

Nitric oxide (NO) is produced by nitric oxide synthases (NOSs) and plays an important role in all levels of reproduction from the brain to the reproductive organs. Recently, it has been discovered that all germ cells and Leydig cells in the cat testis exhibit stage‐dependent nuclear and cytoplasmic endothelial (eNOS) and inducible (iNOS)‐NOS immunoreactivity and cytoplasmic nicotinamide adenine dinucleotide phosphate‐diaphorase (NADPH‐d) reactivity. As a continuation of this finding, in this study, cellular localization of NADPH‐d and immunolocalization and expression of all three NOS isoforms were investigated in the intratesticular (tubuli recti and rete testis), and excurrent ducts (efferent ductules, epididymal duct and vas deferens) of adult cats using histochemistry, immunohistochemistry and western blotting. NADPH‐d activity was found in the midpiece of the spermatozoa tail and epithelial cells of all of ducts, except for nonciliated cells of the efferent ductules. Even though the immunoblotting results revealed similar levels of nNOS, eNOS and iNOS in the caput, corpus and cauda segments of epididymis and the vas deferens, immunostainings showed cell‐specific localization in the efferent ductules and region‐ and cell‐specific localization in the epididymal duct. All of three NOS isoforms were immunolocalized to the nuclear membrane and cytoplasm of the epithelial cells in all ducts, but were found in the tail and the cytoplasmic droplets of spermatozoa. These data suggest that NO/NOS activity might be of importance not only for the functions of the intratesticular and excurrent ducts but also for sperm maturation. Microsc. Res. Tech. 79:192–208, 2016. © 2016 Wiley Periodicals, Inc.     

The application of heat‐deproteinization to the morphological study of cortical bone: A contribution to the knowledge of the osteonal structure

Observation of heat‐deproteinized cortical bone specimens in incident light enabled the high definition documentation of the osteonal pattern of diaphyseal Haversian bone. This prompted a study to compare these images with those revealed by polarized light microscopy, carried out either on decalcified or thin, undecalcified, resin‐embedded sections. Different bone processing methods can reveal structural aspects of the intercellular matrix, depending on the light diffraction mode: birefringency in decalcified sections can be ascribed to the collagen fibrils orientation alone; in undecalcified sections, to both the ordered layout of collagen and the inorganic phase; in the heat‐deproteinized samples, exclusively to the hydroxyapatite crystals aggregation mode. The elemental chemical analysis documented low content of carbon and hydrogen, no detectable levels of nitrogen and significantly higher content of calcium and phosphorus in heat‐deproteinized samples, as compared with dehydrated controls. In both samples, the X‐ray diffraction (XRD) pattern did not show any significant difference in pattern of hydroxyapatite, with no peaks of any possible decomposition phases. Scanning electron microscopic (SEM) morphology of heat‐deproteinized samples could be documented with the fracturing technique facilitated by the bone brittleness. The structure of crystal aggregates, oriented in parallel and with marks of time periods, was documented. Comparative study of deproteinized and undecalcified samples showed that the matrix inorganic phase did not undergo a coarse grain thermal conversion until it reached 500°C, maintaining the original crystals structure and orientation. Incident light stereomicroscopy, combined with SEM analysis of deproteinized bone fractured surfaces, is a new enforceable technique which can be used in morphometric studies to improve the understanding of the osteonal dynamics. Microsc. Res. Tech. 79:691–699, 2016. © 2016 Wiley Periodicals, Inc.     

Visualization of the native shape of bodipy‐labeled DNA in Escherichia coli by correlative microscopy

The native shape and intracellular distribution of newly synthesized DNA was visualized by correlative (light and electron) microscopy in ice embedded whole cells of Escherichia coli. For that purpose, the commercially available modified nucleoside triphosphate named BODIPY® FL‐14‐dUTP was enzymatically incorporated in vivo into the genome of E. coli mutant K12 strain, which cannot synthesize thymine. The successful incorporation of this thymidine analogue was confirmed first by fluorescence microscope, where the cells were stained in the typical for bodipy green color. Later the preselected labeled E. coli were observed by Hilbert Differential Transmission Electron Microscope (HDC TEM) and the distribution of elemental boron (contained in bodipy) was visualized at high‐resolution by an electron spectroscopic imaging (ESI) technique. The practical detection limit of boron was found to be around 5 ～ 10 mmol/kg in area of 0.1 μm², which demonstrated that ESI is a suitable approach to study the cytochemistry and location of labeled nucleic fragments within the cytoplasmic chromosomal area. In addition, the fine cellular fibrous and chromosomal ultrastructures were revealed in situ by combing of phase‐plate HDC TEM and ESI. The obtained results conclude that the correlation between fluorescent microscopy with phase‐plate HDC TEM and ESI is a powerful approach to explore the structural and conformation dynamics of DNA replication machinery in frozen cells close to the living state.     

Curcumin protects against hepatic and renal injuries mediated by inducible nitric oxide synthase during selenium‐induced toxicity in Wistar rats

The aim of this study is to evaluate the effect of curcumin in protecting against selenium‐induced toxicity in liver and kidney of Wistar rats. Light microscopy evaluation of selenium alone administered rats showed liver to be infiltrated with mononuclear cells, vacuolation, necrosis, and pronounced degeneration. Control liver sections showed a regular morphology of parenchymal cells with intact hepatocytes and sinusoids. Kidney from selenium alone administered rats showed vacuolar degeneration changes in the epithelial cells, cellular proliferation with fibrosis, thickening of capillary walls, and glomerular tuft atrophy. Such changes were also observed in rats administered with selenium and curcumin simultaneously and rats administered first with selenium and then curcumin 24 h later. Interestingly, such degenerative changes observed in liver and kidney induced by selenium were not seen in rats that were administered with curcumin first and selenium 24 h later. This clearly suggests the protective nature of curcumin against selenium toxicity. To understand the probable mechanism of action of curcumin, we analyzed inducible nitric oxide synthase (iNOS) expression by immunohistochemistry, and the results showed an increased iNOS expression in selenium‐alone induced liver and kidney. Such high iNOS levels were inhibited in liver and kidney of rats pretreated with curcumin and then with selenium 24 h later. Based on the histological results, it can be concluded that curcumin functions as a protective agent against selenium‐induced toxicity in liver as well as kidney, and this action is probably by the regulatory role of curcumin on iNOS expression. Microsc. Res. Tech., 2010. © 2009 Wiley‐Liss, Inc.     

Aged garlic extract ameliorates the oxidative stress,histomorphological, and ultrastructural changes of cisplatin‐induced nephrotoxicity in adult male rats

Aim: Aged garlic extract (AGE) is a natural dietary substance having different antioxidant free‐radical‐scavenger compounds that ameliorates the toxicity of the oxidative stress. This study aimed to investigate the effect of AGE on cisplatin (CP)‐induced nephrotoxicity in rats. Materials and Methods: Twenty‐four, adult male Wistar albino rats were randomly divided into four groups namely control, AGE‐treated (a single oral dose of 250 mg/kg/day for 21 days), CP‐treated (a single intraperitoneal dose of 7.5 mg/kg on Day 16), and AGE + CP‐treated (AGE at a dose of 250 mg/kg/once daily for 21 days and a single dose of CP of 7.5 mg/kg intraperitoneally on Day 16). Body weight and absolute and relative kidney weights of each rat were calculated. Serum creatinine, uric acid, and urea levels were determined. Level of malondialdehyde and reduced glutathione and activity of superoxide dismutase and catalase of renal tissues were measured. Renal specimens from each rat were prepared for both light and electron microscopic examinations. Results: Interstitial cell infiltration, hemorrhage, glomerular atrophy, necrosis, and tubular degeneration were observed after CP treatment. Superoxide dismutase and catalase activities and glutathione level were significantly decreased and malondialdehyde level was significantly increased in CP‐treated rats compared with AGE + CP‐treated animals. A remarkable improvement in the histopathological and ultrastructural changes induced by CP in renal tissues was observed in AGE + CP‐treated rats. Conclusion: AGE exhibited antioxidant effect that could ameliorate the nephrotoxic effects of CP. Microsc. Res. Tech. 78:452–461, 2015. © 2015 Wiley Periodicals, Inc.