Showing papers on "Intraperitoneal injection published in 2014"

The carcinogenic effect of various multi-walled carbon nanotubes (MWCNTs) after intraperitoneal injection in rats

Abstract: Biological effects of tailor-made multi-walled carbon nanotubes (MWCNTs) without functionalization were investigated in vivo in a two-year carcinogenicity study. In the past, intraperitoneal carcinogenicity studies in rats using biopersistent granular dusts had always been negative, whereas a number of such studies with different asbestos fibers had shown tumor induction. The aim of this study was to identify possible carcinogenic effects of MWCNTs. We compared induced tumors with asbestos-induced mesotheliomas and evaluated their relevance for humans by immunohistochemical methods. A total of 500 male Wistar rats (50 per group) were treated once by intraperitoneal injection with 109 or 5 × 109 WHO carbon nanotubes of one of four different MWCNTs suspended in artificial lung medium, which was also used as negative control. Amosite asbestos (108 WHO fibers) served as positive control. Morbid rats were sacrificed and necropsy comprising all organs was performed. Histopathological classification of tumors and, additionally, immunohistochemistry were conducted for podoplanin, pan-cytokeratin, and vimentin to compare induced tumors with malignant mesotheliomas occurring in humans. Treatments induced tumors in all dose groups, but incidences and times to tumor differed between groups. Most tumors were histologically and immunohistochemically classified as malignant mesotheliomas, revealing a predominantly superficial spread on the serosal surface of the abdominal cavity. Furthermore, most tumors showed invasion of peritoneal organs, especially the diaphragm. All tested MWCNT types caused mesotheliomas. We observed highest frequencies and earliest appearances after treatment with the rather straight MWCNT types A and B. In the MWCNT C groups, first appearances of morbid mesothelioma-bearing rats were only slightly later. Later during the two-year study, we found mesotheliomas also in rats treated with MWCNT D – the most curved type of nanotubes. Malignant mesotheliomas induced by intraperitoneal injection of different MWCNTs and of asbestos were histopathologically and immunohistochemically similar, also compared with mesotheliomas in man, suggesting similar pathogenesis. We showed a carcinogenic effect for all tested MWCNTs. Besides aspect ratio, curvature seems to be an important parameter influencing the carcinogenicity of MWCNTs.

Peripheral administration of tau aggregates triggers intracerebral tauopathy in transgenic mice

Abstract: Soluble tau forms insoluble aggregates following the intracerebral injection of tau inclusions [3, 4]. This is reminiscent of prions, the intracerebral injection of which induces aggregation of cellular prion protein (PrPC) [9]. Multiple routes of administration can transmit prion diseases, with the intracerebral route being more effective than the intraperitoneal route, which is in turn more effective than the oral route [7]. The oral administration of aggregated apolipoprotein A-II [11] and aggregated amyloid protein A (AA) [8] has also been shown to promote systemic amyloidosis. Moreover, intraperitoneally injected aggregated Aβ-containing extracts increased cerebral β-amyloidosis [5]. Here, we report for the first time that the intraperitoneal injection of tau seeds can also induce intracerebral tauopathy. We used homozygous and heterozygous mice transgenic for human mutant P301S tau [2]. In heterozygous mice, tau aggregation forms later than in homozygous mice, and heterozygous mice live longer than their homozygous counterparts (15 months as opposed to 6 months). Brainstem extracts from 6-month-old homozygous P301S tau transgenic mice (prepared and analysed as previously described [3]) were injected into the peritoneal cavity of 3-month-old heterozygous mice, an age at which they lack tau deposits. The mice were analysed 9 months after the final injection. The number of Gallyas silver-positive cells was assessed using a scanning light microscope. Maps of entire brain sections, along with the annotation of the brain outline and artificially altered areas, were used for the counting of black Gallyas-positive and blue haematoxylin-positive structures (Supplementary Fig. 4). Gallyas per cell nucleus (G/N) ratios were counted, thus reflecting the number of silver-positive cells. G/N ratios were calculated per field of view (20× objective, 661.5 × 372.1 μm). Gauss-blurred ratiometric images (sigma = 70 pixels, pixel edge length = 3.45 μm) were superimposed for the control and experimental groups. Subsequently, a pixel-wise Student’s t test was performed and the probability (p) values were plotted, with the statistically significant portion (p < 0.05) being represented as a colour map (Fig. 2). Our ratiometric approach (G/N) only compares equivalent brain regions to each other. Thus, despite anatomical differences in densities of nuclei, our comparative maps remain unbiased. Fig. 2 Gallyas silver-positive structures per cell nucleus (G/N ratios) in non-injected controls (CO) and in mice injected with aggregated tau-rich homogenate either intraperitoneally (IP) or intracerebrally (IC). a Left panel colour map representing average ... Although groups of mice injected intraperitoneally (IP) with brainstem homogenate from either P301S tau transgenic mice (with tau filaments) or control (CO) mice (without tau filaments) formed Gallyas-positive structures in the brain, there were also significant differences between the two groups (Fig. 1). G/N ratios (Fig. 2a, left panel) showed a marked increase in the number of silver-positive structures in the brainstem and neocortex of mice IP injected with P301S brainstem extract. The probability map (Fig. 2a, right panel) showed large differences in secondary motor cortex, ventral orbital cortex and olfactory nucleus. More ventral regions, including pallidum and lateral preoptic area, showed more silver-positive inclusions in P301S brainstem-injected mice, as did lateral habenular nucleus, thalamic nucleus, pretectal nucleus and mesencephalic reticular formation. In the brainstem, pontine reticular nucleus, gigantocellular reticular nucleus, median vestibular nucleus and solitary tract showed the largest increase in Gallyas-positive inclusions (Supplementary Fig. 2). The hippocampus remained unaffected. At the age of analysis (12 months), brains of heterozygous P301S mice develop neuronal inclusions made of hyperphosphorylated (AT8 positive, pretangles) and aggregated tau (AT100- and Gallyas silver-positive, classical tangles). Both AT8 and AT100 tau immunoreactivity (Supplementary Fig. 3) as well as silver staining in IP-injected mice were increased compared to non-injected mice, but the pattern (brain distribution and morphology) of tau pathology remained unchanged. Age-matched wild-type mice injected intraperitoneally with P301S brainstem extract failed to develop cerebral tauopathy. Fig. 1 Intraperitoneal injection of aggregated tau-rich homogenate increases the number of silver-positive tau inclusions in brain. Gallyas-Braak staining of the brainstem of 12-month-old mice heterozygous for transgenic human mutant P301S tau showed an increase ... We previously showed that the intracerebral injection of silver-positive brainstem extract from mice homozygous for human mutant P301S tau into mice transgenic for human wild-type tau (line ALZ17) induced the formation of silver-positive inclusions [3]. To investigate if this is also true of another mouse model of tauopathy, we injected brainstem extract from homozygous mice transgenic for human mutant P301S tau into the hippocampus and overlying cerebral cortex of 3-month-old heterozygous mice and compared the relative efficiency of intracerebral and intraperitoneal injections after 9 months. Following intracerebral injection, numerous Gallyas-positive structures were present in the CA1 region of the hippocampus and in the secondary motor cortex (Supplementary Fig. 1A). No silver-positive structures were present in these regions following the intraperitoneal injection of control extracts. Heat maps revealed a significant increase in the G/N ratio in the hippocampus following the intracerebral injection of P301S brainstem extract into heterozygous transgenic P301S tau mice (Fig. 2b, left panel). The p maps showed statistically significant differences between groups in the hippocampal area, which was a site of intracerebral injection (Fig. 2b, right panel; Supplementary Fig. 1B). Moreover, the G/N ratio was increased more in brainstem and neocortex following intracerebral than after intraperitoneal injections. Heart, lungs, liver, spleen and kidneys from injected heterozygous P301S tau mice were all silver-negative and tau-negative. No sign of inflammation was detected in either the brain or the periphery. These findings demonstrate that aggregated tau seeds, such as prions and Aβ aggregates, can reach the central nervous system from the periphery. The underlying mechanisms remain to be determined. The replication of peripherally applied prions and their translocation to the brain are dependent on haematopoietic and stromal immune cells, together with the sympathetic innervation of abdominal lymphoid organs [1]. In mouse models of AA-amyloidosis, peripheral blood monocytes transported the aggregates [10]. It therefore appears that amyloid seeds can be carried by blood cells. This may also be true of aggregated tau. Seeding may require the presence of unfolded monomers [6]. It remains to be determined if tau seeds are able to replicate in the peripheral nervous system. Their transport to the brain may not require endogenous tau, which is not expressed by lymphoreticular cells. This is unlike prion propagation, which depends on the expression of PrPC by stromal and haematopoietic cells. Because the central nervous system is affected in clinical tauopathy, the intracerebral route of seed delivery could be expected to be more effective than the intraperitoneal route. This was the case here, where more silver-positive structures formed in brainstem, hippocampus and neocortex following the intracerebral injection of 5 μl brainstem homogenate (0.19 μg of τ/μl of homogenate) than after the intraperitoneal injection of 200 μl homogenate (0.19 μg of τ/μl of homogenate). It follows that, like prion diseases, tauopathies can be seeded in the brain by tau aggregates delivered peripherally, although the intraperitoneal administration was less effective than the intracerebral route. These findings underscore the urgent need for additional work on the aetiology and pathogenesis of tauopathies.

Melatonin ameliorates oxidative stress and reproductive toxicity induced by cyclophosphamide in male mice.

Abstract: The present study evaluated the efficacy of melatonin in cyclophosphamide (CP)-induced testicular injury, lipid peroxidative damage, and antioxidant enzymes status of the mice testis on the basis of biochemical and histological studies. Mice were pretreated with four different doses of melatonin (2.5, 5, 10, and, 20 mg/kg by body weight (b.w.)) via intraperitoneal injection for five consecutive days followed by injection with CP (200 mg/kg b.w.) 1 h after the last injection of melatonin on the 5th day. After 24 h, mice were euthanized, testes were immediately removed, and biochemical and histological studies were conducted. Treatment with melatonin significantly mitigates lipid peroxidation, superoxide dismutase, and catalase activity and the level of reduced glutathione content abnormality induced by CP in mice testis. Histological examination clearly demonstrates that pretreatment of melatonin prevented CP-induced spermatogenesis toxicity and spermatogenic cells reduction in mice testis. The protective effect of melatonin is likely due to the antioxidative properties of the indolamine existed in the chemical structure. Because melatonin is a safe, natural compound, it could be used concomitantly as a supplement to protect people undergoing chemotherapy against reproductive toxicity.

Intraperitoneal injection of in vitro expanded Vγ9Vδ2 T cells together with zoledronate for the treatment of malignant ascites due to gastric cancer

Abstract: Malignant ascites caused by peritoneal dissemination of gastric cancer is chemotherapy-resistant and associated with poor prognosis. We conducted a pilot study to evaluate the safety of weekly intraperitoneal injections of in vitro expanded Vγ9Vδ2 T cells together with zoledronate for the treatment of such malignant ascites. Patient peripheral blood mononuclear cells were stimulated with zoledronate (5 μmol/L) and interleukin-2 (1000 IU/mL). After 14 days culture, Vγ9Vδ2 T-cells were harvested and administered intraperitoneally in four weekly infusions. The day before T-cell injection, patients received zoledronate (1 mg) to sensitize their tumor cells to Vγ9Vδ2 T-cell recognition. Seven patients were enrolled in this study. The number of Vγ9Vδ2 T-cells in each injection ranged from 0.6 to 69.8 × 108 (median 59.0 × 108). There were no severe adverse events related to the therapy. Intraperitoneal injection of Vγ9Vδ2 T cells allows them access to the tumor cells in the peritoneal cavity. The number of tumor cells in the ascites was significantly reduced even after the first round of therapy and remained substantially lower over the course of treatment. IFN-γ was detected in the ascites on treatment. Computed tomography revealed a significant reduction in volume of ascites in two of seven patients. Thus, injection of these antitumor Vγ9Vδ2 T-cells can result in local control of malignant ascites in patients for whom no standard therapy apart from paracentesis is available. Adoptively transferred Vγ9Vδ2 T-cells do indeed recognize tumor cells and exert antitumor effector activity in vivo, when they access to the tumor cells.

In vivo effects of synthetic cannabinoids JWH-018 and JWH-073 and phytocannabinoid Δ9-THC in mice: Inhalation versus intraperitoneal injection

Abstract: Human users of synthetic cannabinoids (SCBs) JWH-018 and JWH-073 typically smoke these drugs, but preclinical studies usually rely on injection for drug delivery. We used the cannabinoid tetrad and drug discrimination to compare in vivo effects of inhaled drugs with injected doses of these two SCBs, as well as with the phytocannabinoid Δ9-tetrahydrocannabinol (Δ9-THC). Mice inhaled various doses of Δ9-THC, JWH-018 or JWH-073, or were injected intraperitoneally (IP) with these same compounds. Rectal temperature, tail flick latency in response to radiant heat, horizontal bar catalepsy, and suppression of locomotor activity were assessed in each animal. In separate studies, mice were trained to discriminate Δ9-THC (IP) from saline, and tests were performed with inhaled or injected doses of the SCBs. Both SCBs elicited Δ9-THC-like effects across both routes of administration, and effects following inhalation were attenuated by pretreatment with the CB1 antagonist/inverse agonist rimonabant. No cataleptic effects were observed following inhalation, but all compounds induced catalepsy following injection. Injected JWH-018 and JWH-073 fully substituted for Δ9-THC, but substitution was partial (JWH-073) or required relatively higher doses (JWH-018) when drugs were inhaled. These studies demonstrate that the SCBs JWH-018 and JWH-073 elicit dose-dependent, CB1 receptor-mediated Δ9-THC-like effects in mice when delivered via inhalation or via injection. Across these routes of administration, differences in cataleptic effects and, perhaps, discriminative stimulus effects, may implicate the involvement of active metabolites of these compounds.

Hepatoprotective effects of zataria multiflora ethanolic extract on liver toxicity induced by cyclophosphamide in mice.

Abstract: Although cyclophosphamide (CP), an alkylating agent, has been extensively used in chemotherapy, it possesses a wide spectrum of adverse effects including hepatotoxicity. This study was aimed to evaluate the protective effects of Zataria multiflora against hepatic damage induced by CP in mice. Mice were orally (gavages) pretreated with the ethanolic extract aerial parts of Zataria at doses of 50, 100, 200, or 400 mg/kg for 7 consecutive days before a single intraperitoneal injection of 200 mg/kg CP. After 24 h, animals were anesthetized, blood samples and hepatic tissues were collected and used for biochemical and histological examination. Serum levels of hepatic markers were significantly increased after only CP treated animals but restored in Zataria pretreated groups. A single dose of CP administration also markedly induced abnormality in the levels of several biomarkers associated with oxidative stress in liver tissues homogenates. However, pretreatment with Zataria significantly inhibited the abnormality of antioxidant enzymes defense system in the liver tissues. In addition, histopathological studies proved that CP causes damage to the liver, and this was evidenced by the induced dilated and congested sinusoidal space, lymphocytic infiltration between hepatocytes, portal space with moderate to severe inflammation and necrotic hepatocyte with absence of nuclei. Zataria effectively protected animals against CP-induced hepatic tissue damages. Our results reveal that Zataria produces a potent hepatoprotective role and could be a potent candidate to use concomitantly as a supplement agent against hepatotoxicity of CP for the patients undergoing chemotherapy.

Adiponectin agonist ADP355 attenuates CCl4-induced liver fibrosis in mice.

Abstract: Liver fibrosis is a growing global health problem characterized by excess deposition of fibrillar collagen, and activation of hepatic stellate cells (HSCs). Adiponectin is known to possess anti-fibrotic properties; however a high physiological concentration and multiple forms circulating in blood prohibit clinical use. Recently, an adiponectin-like small synthetic peptide agonist (ADP355: H-DAsn-Ile-Pro-Nva-Leu-Tyr-DSer-Phe-Ala-DSer-NH2) was synthesized for the treatment of murine breast cancer. The present study was designed to evaluate the efficacy of ADP355 as an anti-fibrotic agent in the in vivo carbon tetrachloride (CCl4)-induced liver fibrosis model. Liver fibrosis was induced in eight-week old male C57BL/6J mice by CCl4-gavage every other day for four weeks before injection of a nanoparticle-conjugated with ADP355 (nano-ADP355). Control gold nanoparticles and nano-ADP355 were administered by intraperitoneal injection for two weeks along with CCl4-gavage. All mice were sacrificed after 6 weeks, and serum and liver tissue were collected for biochemical, histopathologic and molecular analyses. Biochemical studies suggested ADP355 treatment attenuates liver fibrosis, determined by reduction of serum aspartate aminotransferase (AST), alanine aminotransferase ALT) and hydroxyproline. Histopathology revealed chronic CCl4-treatment results in significant fibrosis, while ADP355 treatment induced significantly reversed fibrosis. Key markers for fibrogenesis–α-smooth muscle actin (α-SMA), transforming growth factor-beta1 (TGF-β1), connective tissue growth factor (CTGF), and the tissue inhibitor of metalloproteinase I (TIMP1) were also markedly attenuated. Conversely, liver lysates from ADP355 treated mice increased phosphorylation of both endothelial nitric oxide synthase (eNOS) and AMPK while AKT phosphorylation was diminished. These findings suggest ADP355 is a potent anti-fibrotic agent that can be an effective intervention against liver fibrosis.

SIRT1 activator (SRT1720) improves the follicle reserve and prolongs the ovarian lifespan of diet-induced obesity in female mice via activating SIRT1 and suppressing mTOR signaling

Abstract: The prevalence of obesity is increasing worldwide and significantly affects fertility and reproduction in both men and women. Our recent study has shown that excess body fat accelerates ovarian follicle development and follicle loss in rats. The aim of the present study is to explore the effect of SIRT1 activator SRT1720 on the reserve of ovarian follicle pool and ovarian lifespan of obese mice and the underlying mechanism associated with SIRT1 and mTOR signaling. Adult female Kunming mice (n = 36) were randomly divided into three groups: the normal control (NC) group (n = 8), the caloric restriction (CR) group (fed 70% food of the NC group, n = 8) and the high-fat diet (HF) group (fed a rodent chow containing 20% fat, n = 20). After 4 months, the HF mice were further randomly divided into three groups: the control high-fat diet (CHF, n = 8) group (treated every day with an intraperitoneal injection of vehicle), the SRT1720 (SRT, n = 6) group (treated every other day with an intraperitoneal injection of SRT1720 (50 mg/kg)), the SRT1720 and nicotinamide (NAM, n = 6) group (treated every other day with an intraperitoneal injection of SRT1720 (50 mg/kg) and every day with an intraperitoneal injection of nicotinamide (100 mg/kg)). After 6 weeks of treatment, ovaries were harvested for histological and Western blotting analyses. The body weight, ovary weight and visceral fat in the SRT group were significantly lower than those in the CHF group at the end of treatment. Histological analysis showed that the SRT mice had significantly greater number and percentage of primordial follicles, but lower number and percentage of corpora lutea and atretic follicles than the CHF mice and NAM mice. Western blot analysis demonstrated that the levels of SIRT1, SIRT6, FOXO3a and NRF-1 protein expression significantly increased in the ovaries of SRT mice, whereas those of mTORC1, p-mTOR, p-p70S6K, NFκB and p53 decreased compared to the CHF and NAM mice. Our study suggests that SRT1720 may improve the follicle pool reserve in HF diet-induced obese female mice via activating SIRT1 signaling and suppressing mTOR signaling, thus extending the ovarian lifespan.

P2X7 receptor inhibition interrupts the progression of seizures in immature rats and reduces hippocampal damage.

Abstract: AIMS: Early-life seizures, particularly when prolonged, may be harmful to the brain Current pharmacotherapy is often ineffective; therefore, novel neuro- and/or glio-transmitter systems should be explored for targeting The P2X7 receptor is a cation-permeable channel with trophic and excitability effects on neurons and glia which is activated by high amounts of ATP that may be released in the setting of injury after severe seizures Here, we tested the effects of A-438079, a potent and selective P2X7 receptor antagonist in a lesional model of early-life status epilepticus METHODS: Seizures were induced by intra-amygdala kainic acid in 10-day-old rat pups Electrographic seizure severity, changes to P2X7 receptor expression, inflammatory responses and histological effects were evaluated RESULTS: Seizures induced by intra-amygdala kainic acid increased levels of P2X7 receptor protein and interleukin-1beta and caused significant cell death within the ipsilateral hippocampus A-438079 rapidly reached the brain following systemic injection in P10 rats Intraperitoneal injection of A-438079 (5 and 15 mg/kg) 60 min after triggering seizures reduced seizure severity and neuronal death within the hippocampus A-438079 had superior neuroprotective effects compared with an equally seizure-suppressive dose of phenobarbital (25 mg/kg) CONCLUSIONS: These results suggest P2X7 receptor antagonists may be suitable as frontline or adjunctive treatments of pediatric status epilepticus or other early-life seizures, particularly when associated with brain damage

Anti-Inflammatory Effect of Momordica Charantia in Sepsis Mice

Abstract: Wild bitter gourd (Momordica charantia L. var. abbreviate Seringe), a common vegetable in Asia, is used in traditional medicine to treat various diseases, including inflammation. Extant literature indicates that wild bitter gourds have components that activate PPARα and PPARγ. This research probed the influence of adding wild bitter gourd to diets on inflammation responses in mice with sepsis induced by intraperitoneal injection of LPS. Male BALB/c mice were divided normal, sepsis, positive control, and three experimental groups. The latter ate diets with low (1%), moderate (2%), and high (10%) ratios of wild bitter gourd lyophilized powder. Before mice were sacrificed, with the exception of the normal group, intraperitoneal injection of LPS induced sepsis in each group; positive control group was injected with LPS after PDTC. This experiment revealed starkly lower weights in groups with added wild bitter gourd than those of the remaining groups. Blood lipids (TG, cholesterol, and NEFA) were also lower in comparison to the sepsis group, and blood glucose concentrations recovered and approached normal levels. Blood biochemistry values related to inflammation reactions indicated GOT, GPT, C-RP, and NO concentrations of groups with added wild bitter gourd were all lower than those of the sepsis group. Secretion levels of the spleen pro-inflammatory cytokines IL-1, IL-6, and TNF-α tallied significantly lower in comparison to the sepsis group, whereas secretion levels of IL-10 anti-inflammatory cytokine increased. Expression level of proteins NF-κB, iNOS, and COX-2 were significantly inhibited. Results indicate wild bitter gourd in diets promoted lipid metabolism, reducing fat accumulation, and improving low blood glucose in sepsis. Addition of wild bitter gourd can reduce inflammation biochemical markers or indicators and pro-inflammatory cytokines in the body, hence improving the inflammation responses in mice with sepsis.

Ultrasound-mediated destruction of LHRHa-targeted and paclitaxel-loaded lipid microbubbles for the treatment of intraperitoneal ovarian cancer xenografts.

Abstract: Ultrasound-targeted microbubble destruction (UTMD) is a promising technique to facilitate the delivery of chemotherapy in cancer treatment. However, the process typically uses nonspecific microbubbles, leading to low tumor-to-normal tissue uptake ratio and adverse side effects. In this study, we synthesized the LHRH receptor-targeted and paclitaxel (PTX)-loaded lipid microbubbles (TPLMBs) for tumor-specific binding and enhanced therapeutic effect at the tumor site. An ovarian cancer xenograft model was established by injecting A2780/DDP cells intraperitoneally in BALB/c nude mice. Microscopic imaging of tumor sections after intraperitoneal injection of TPLMBs showed effective binding of the microbubbles with cancer cells. Ultrasound mediated destruction of the intraperitoneally injected TPLMBs yielded a superior therapeutic outcome in comparison with other treatment options. Immunohistochemical analyses of the dissected tumor tissue further confirmed the increased tumor apoptosis and reduced angiogenesis. Our experiment suggests that ultrasound-mediated intraperitoneal administration of the targeted drug-loaded microbubbles may be a useful method for the treatment of ovarian cancer.

Orally administered lycopene attenuates diethylnitrosamine-induced hepatocarcinogenesis in rats by modulating Nrf-2/HO-1 and Akt/mTOR pathways.

Abstract: Hepatocarcinogenesis is one of the most prevalent and lethal cancers. We studied the mechanisms underlying the inhibition of diethylnitrosamine (DEN)-induced hepatocarcinogenesis by lycopene in rats. Hepatocarcinogenesis was induced by an intraperitoneal injection of DEN followed by promotion with phenobarbital for 24 successive wk. The rats were given lycopene (20 mg/kg body weight) 3 times a week orally for 4 wk prior to initiation, and the treatment was continued for 24 consecutive wk. Lycopene reduced incidence, number, size, and volume of hepatic nodules. Serum alanine transaminase, aspartate aminotransferase, total bilirubin, and malondialdehyde (MDA) considerably increased and hepatic antioxidant enzymes (catalase, superoxide dismutase, glutathione peroxidase) and glutathione decreased in DEN-treated rats when compared with the control group. Lycopene significantly reversed these biochemical changes and increased the expression of NF-E-2-related factor-2)/heme oxygenase-1, and it decreased NF-κB/cy...

Ferulic acid in the treatment of post-diabetes testicular damage: relevance to the down regulation of apoptosis correlates with antioxidant status via modulation of TGF-β1, IL-1β and Akt signalling

Abstract: The aim of this study was to investigate the protective effect of ferulic acid at different doses (50 mg kg(-1) alternative day and 50 mg kg(-1) daily) on the streptozotocin (STZ)-induced post-diabetes rat testicular damage. Diabetes was induced by a single intraperitoneal injection of STZ (50 mg/kg). Rats treated with ferulic acid were given once a day orally for 10 weeks, starting 3 days after STZ injection. Testis tissue and blood samples were collected for investigating biochemical analysis, antioxidant status, sperm parameters, and histopathological, immunohistochemical and apoptotic studies. Treatment with ferulic acid to diabetic rats significantly improved the body weight, testis weight, serum insulin level, serum testosterone level and sperm parameters (viability, motility and count). Histopathological study also revealed that ferulic acid-treated diabetic rats showed an improved histological appearance. Our data indicated that significant reduction in the activity of apoptosis by using terminal deoxyuridine triphosphate nick end-labelling and reduced expression of transforming growth factor-β1 and interleukin-1β in the testis tissue of ferulic acid-treated diabetic rats. Conversely, it was also revealed that ferulic acid-treated diabetic rats markedly enhanced the serine/threonine protein kinase protein expression in the testis tissue. Our result suggests that ferulic acid inhibits testicular damage in diabetic rats by declining oxidative stress.

Acute and chronic administration of gold nanoparticles cause DNA damage in the cerebral cortex of adult rats

Abstract: The use of gold nanoparticles is increasing in medicine; however, their toxic effects remain to be elucidated. Studies show that gold nanoparticles can cross the blood–brain barrier, as well as accumulate in the brain. Therefore, this study was undertaken to better understand the effects of gold nanoparticles on rat brains. DNA damage parameters were evaluated in the cerebral cortex of adult rats submitted to acute and chronic administration of gold nanoparticles of two different diameters: 10 and 30 nm. During acute administration, adult rats received a single intraperitoneal injection of either gold nanoparticles or saline solution. During chronic administration, adult rats received a daily single injection for 28 days of the same gold nanoparticles or saline solution. Twenty-four hours after either single (acute) or last injection (chronic), the rats were euthanized by decapitation, their brains removed, and the cerebral cortices isolated for evaluation of DNA damage parameters. Our study showed that acute administration of gold nanoparticles in adult rats presented higher levels of damage frequency and damage index in their DNA compared to the control group. It was also observed that gold nanoparticles of 30 nm presented higher levels of damage frequency and damage index in the DNA compared to the 10 nm ones. When comparing the effects of chronic administration of gold nanoparticles of 10 and 30 nm, we observed that occurred significant different index and frequency damage, comparing with control group. However, there is no difference between the 10 and 30 nm groups in the levels of DNA damage for both parameters of the Comet assay. Results suggest that gold nanoparticles for both sizes cause DNA damage for chronic as well as acute treatments, although a higher damage was observed for the chronic one.

Intraperitoneal injection improves the uptake of nanoparticle-labeled high-density lipoprotein to atherosclerotic plaques compared with intravenous injection: a multimodal imaging study in ApoE knockout mice

Abstract: Background— The aim of this study was to assess whether high-density lipoprotein (HDL) labeled with superparamagnetic iron oxide nanoparticles (SPIOs) and quantum dots was able to detect atherosclerotic lesions in mice after intravenous and intraperitoneal injection by multimodal imaging. Methods and Results— Nanoparticle-labeled HDLs (NP-HDLs) were characterized in vitro by dynamic light scattering and size exclusion chromatography with subsequent cholesterol and fluorescence measurements. For biodistribution and blood clearance studies, NP-HDLSPIOs radiolabeled with 59Fe (NP-HDL59Fe-SPIOs) were injected intravenously or intraperitoneally into ApoE knockout mice (n=6), and radioactivity was measured using a gamma counter. NP-HDL accumulation within atherosclerotic plaques in vivo and ex vivo was estimated by MRI at 7 Tesla, ex vivo confocal fluorescence microscopy, x-ray fluorescence microscopy, and histological analysis (n=3). Statistical analyses were performed using a 2-tailed Student t -test. In vitro characterization of NP-HDL confirmed properties similar to endogenous HDL. Blood concentration time curves showed a biexponential decrease for the intravenous injection, whereas a slow increase followed by a steady state was noted for intraperitoneal injection. Radioactivity measurements showed predominant accumulation in the liver and spleen after both application approaches. NP-HDL59Fe-SPIOs uptake into atherosclerotic plaques increased significantly after intraperitoneal compared with intravenous injection ( P <0.01). In vivo MRI showed an increased uptake of NP-HDL into atherosclerotic lesions after intraperitoneal injection, which was confirmed by ex vivo MRI, x-ray fluorescence microscopy, confocal fluorescence microscopy, and histological analysis. Conclusions— In vivo MRI and ex vivo multimodal imaging of atherosclerotic plaque using NP-HDL is feasible, and intraperitoneal application improves the uptake within vessel wall lesions compared with intravenous injection.

Bone mesenchymal stem cell transplantation via four routes for the treatment of acute liver failure in rats.

Abstract: In the present study, we assessed the efficiency of four BMSC transplantation methods as a therapy for liver failure. A rat model (80 Sprague-Dawley rats) of D-galactosamine (D-gal)/lipopolysaccharide (LPS)-induced acute liver failure (ALF) was established and the rats were divided into 5 groups: a hepatic artery injection group, a portal vein injection group, a vena caudalis injection group, an intraperitoneal injection group and a control group (16 per group). Following transplantation, the liver tissue and blood samples were collected on days 1, 3 and 7, we detected the EdU (5-ethynyl-2′-deoxyuridine)-labeled cells homing to the liver tissue and assessed the proliferating cell nuclear antigen (PCNA) and cysteine-containing aspartate-specific protease (caspase)-3 expression in the liver tissue and detected the levels of stromal cell-derived factor 1 (SDF-1) and hepatocyte growth factor (HGF) in the liver tissues. Compared with the control group, the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and damage to the liver tissue in the hepatic artery group, the portal vein group and the vena caudalis group improved in vivo. The expression of PCNA and HGF in the liver was higher and caspase-3 expression was lower in the hepatic artery injection group, the portal vein injection group and the vena caudalis injection group than that in the intraperitoneal injection and control groups. The EdU-labeled BMSCs were only observed homing to the liver tissue in these three groups. However, no significant differences were observed between these three groups. Liver function in the rats with ALF was improved following BMSC transplantation via 3 endovascular implantation methods (through the hepatic artery, portal vein and vena caudalis). These 3 methods were effective in transplanting BMSCs for the treatment of ALF. However, the selection of blood vessel in the implantation pathway does not affect the transplantation outcome. Transplantation via intraperitoneal injection showed no therapeutic effect in our animal experiments.

Establishment of a Rat Model of Type II Diabetic Neuropathic Pain

Abstract: Objective To establish a rat model of type II diabetic neuropathic pain. Methods Sixty Sprague Dawley rats were randomly divided into two groups: group A (N = 10) was fed a normal diet, and group B (N = 50) was fed a high-fat and high-sugar diet. After 8 weeks, the body weight of all rats was recorded, and rats in both groups had their fasting plasma glucose, insulin concentration, and insulin sensitivity index measured and calculated. Subsequently, the rats in group B were randomly divided into three subgroups that were each given different doses of streptozotocin (STZ) by a single intraperitoneal injection (subgroup B1 received 30 mg/kg, subgroup B2 received 35 mg/kg, and subgroup B3 40 mg/kg). Two weeks after the STZ injection, the four groups of rats had their insulin sensitivity index, mechanical withdrawal threshold, and thermal withdrawal latency assessed, allowing us to establish a rat model of type II diabetic neuropathic pain and to determine the optimum dose of STZ. Four weeks after STZ injection (2 weeks after the model was established), the pain threshold was measured in the rats in group A and the group treated with the most effective STZ dose. We also measured the expression of phosphorylated extracellular signal-regulated kinase (p-ERK), phosphorylated cyclic AMP response element-binding protein (p-CREB), and phosphorylated N -methyl d-aspartate receptor subtype B (p-NR2B) in the dorsal root ganglion (DRG) and spinal cord dorsal horn regions, which are closely related to neuropathic pain, and also recorded the TTX-R sodium currents in the acutely isolated DRG neurons. Results After 8 weeks of a high-fat, high-sugar diet, the body weight of the rats in group B was significantly increased. Although the fasting blood glucose levels did not change significantly, the fasting insulin levels were slightly elevated, and the insulin sensitivity index was significantly reduced. Two weeks after STZ injection, the blood glucose levels of the rats in subgroup B1 were elevated but did not remain so for a prolonged period. In contrast, the rats in subgroup B3 had elevated blood glucose that was accompanied by a high mortality rate, while the blood glucose levels of the rats in subgroup B2 were moderately elevated and relatively stable. In addition, the pain threshold was significantly decreased ( P < 0.05), and the mortality was low in this group. Because of this, the dose of STZ that was used in group B2 was considered the most effective dose of STZ for induction of diabetes. Four weeks after STZ injection, the pain threshold in the rats of group B2 was still significantly decreased, and the expression of p-ERK, p-CREB, and p-NR2B in the dorsal root ganglion (DRG) and spinal cord dorsal horn was significantly increased. The tetrodotoxin-resistant sodium current density in DRG neurons was also significantly elevated ( P < 0.05). Conclusions A rat model of type II diabetic neuropathic pain can be established by feeding rats a high-fat, high-sugar diet for 8 weeks, in combination with intraperitoneal injection of 35 mg/kg STZ. This model can be stably maintained for at least 2 weeks.

Subretinal delivery of AAV2-mediated human erythropoietin gene is protective and safe in experimental diabetic retinopathy.

Abstract: Purpose We studied and developed a gene-based intraocular erythropoietin (EPO) therapy for diabetic retinopathy (DR), by which the applicability of neuroprotective therapy with favorable safety profile is attempted. Methods Hematocrit (Hct) was measured in C57BL/6 mice after intramuscular injection of AAV2-CMV-hEPO virus. Diabetes was induced by intraperitoneal injection of streptozotocin in Sprague-Dawley (SD) rats. Subretinal or intravitreal injection was performed in SD rats and Dark Agouti (DA) rats. The human EPO (hEPO) concentration was measured with ELISA. Blood-retinal barrier (BRB) breakdown was measured with Evans blue permeation. Retinal function was evaluated with electroretinography (ERG). Retinal cell apoptosis was detected with TUNEL. Retinal thickness and cell counts were examined by light microscopy. Retinal vascular changes were evaluated with fundus fluorescein angiography (FFA) and confocal microscopy. Results The serum hEPO was elevated 2 weeks after AAV2-CMV-hEPO virus injection, and Hct began to increase after 4 weeks. After subretinal injection, hEPO expressions in aqueous humor, vitreous, and retina followed a dose- and time-dependent manner. In the AAV2-CMV-hEPO-treated diabetic group, BRB was maintained, and retinal cell apoptosis was significantly reduced. The ERG results showed that the retinal function remained unchanged for at least one year after subretinal injection of AAV2-CMV-hEPO virus. Long-term expression of hEPO following subretinal injection of AAV2-CMV-hEPO virus did not induce neovascularization in retina and choroid. Conclusions The AAV2-CMV-hEPO gene therapy is safe, and it exerts long-term protective effects on diabetic retinas. Thus, the gene therapy by using AAV2-CMV-hEPO for DR is feasible.

A Novel Angiogenesis Inhibitor Bevacizumab Induces Apoptosis in the Rat Endometriosis Model

Abstract: Our aim was to investigate the effects of anti-vascular endothelial growth factor (anti-VEGF) antibody Bevacizumab on endometrial explants and on apoptotic gene expression levels in the rat endometriosis model. Endometriotic implants were surgically formed, and rats treated with (i) 1 mg/kg single subcutaneous injection of depot leuprolide acetate; (ii) 2.5 mg/kg of single intaperitoneal injection of bevacizumab; (iii) intraperitoneal injection of saline. Histopathologic scores and adhesion scores of endometriotic foci and levels of Bcl-2-associated X protein (Bax), Cytochrome c (Cyt-c), B-cell lymphoma/leukemia 2 (Bcl-2) and B-cell lymphoma-extra large (Bcl-xl) mRNA gene expressions of endometriotic foci. Bevacizumab treatment decreased the endometriotic explant size compared with control. Bevacizumab-treated rats had lower total adhesion scores when compared with the control group. Semi-quantitative evaluation of the persistence of endometrial epithelial cells in the explants showed a lower score in gonadotropin-releasing hormone (GnRH) agonist-treated rats compared with control rats. In Bevacizumab increased expression of Bax 3.1-fold, Cyt-c 1.3-fold and decreased expression of Bcl-2 0.4-fold, Bcl-xl 0.8-fold compared with the control group. The GnRH agonist increased expression of Bax 3.0 fold, Cyt-c 1.3 fold and decreased expression of Bcl-2 0.4-fold, Bcl-xl 0.8-fold, compared with the control group. This study suggests that a novel angiogenesis inhibitor, anti-VEGF antibody bevacizumab is as effective as GnRH agonist in the regression of the endometriotic lesions in rat endometriosis model. One possible mechanism of this effect is the induction of apoptosis.

The thioredoxin reductase-1 inhibitor aurothioglucose attenuates lung injury and improves survival in a murine model of acute respiratory distress syndrome.

Abstract: Aims: Inflammation and oxygen toxicity increase free radical production and contribute to the development of acute respiratory distress syndrome (ARDS), which is a significant cause of morbidity and mortality in intensive care patients. We have previously reported increased glutathione (GSH) levels in lung epithelial cells in vitro and attenuated adult murine hyperoxic lung injury in vivo after pharmacological thioredoxin reductase-1 (TrxR1) inhibition. Using a murine ARDS model, we tested the hypothesis that aurothioglucose (ATG) treatment increases pulmonary GSH levels, attenuates lung injury, and decreases mortality in a GSH-dependent manner. Results: Adult mice received a single intratracheal dose of 0.375 μg/g lipopolysaccharide (LPS) 12 h before a single intraperitoneal injection of 25 mg/kg ATG. Control mice received intratracheal and/or intraperitoneal saline. Mice were then exposed to room air or hyperoxia (>95% O2). Lung injury was assessed by bronchoalveolar lavage protein concentratio...

HET0016, a selective inhibitor of 20-HETE synthesis, decreases pro-angiogenic factors and inhibits growth of triple negative breast cancer in mice.

Abstract: A selective inhibitor of 20-HETE synthesis, HET0016, has been reported to inhibit angiogenesis. 20-HETE has been known as a second mitogenic messenger of angiogenesis inducing growth factors. HET0016 effects were analyzed on MDA-MB-231 derived breast cancer in mouse and in vitro cell line. MDA-MB-231 tumor cells were implanted in animals’ right flank and randomly assigned to early (1 and 2), starting treatments on day 0, or delayed groups (3 and 4) on day 8 after implantation of tumor. Animals received HET0016 (10 mg/kg) treatment via intraperitoneal injection for 5 days/week for either 3 or 4 weeks. Control group received vehicle treatment. Tumor sizes were measured on days 7, 14, 21, and 28 and the animals were euthanized on day 22 and 29. Proteins were extracted from the whole tumor and from cells treated with 10 µM HET0016 for 4 and 24 hrs. Protein array kits of 20 different cytokines/factors were used. ELISA was performed to observe the HIF-1α and MMP-2 protein expression. Other markers were confirmed by IHC. HET0016 significantly inhibited tumor growth in all treatment groups at all-time points compared to control (p<0.05). Tumor growth was completely inhibited on three of ten animals on early treatment group. Treatment groups showed significantly lower expression of pro-angiogenic factors compared to control at 21 days; however, there was no significant difference in HIF-1α expression after treatments. Similar results were found in vitro at 24 hrs of HET0016 treatment. After 28 days, significant increase of angiogenin, angiopoietin-1/2, EGF-R and IGF-1 pro-angiogenic factors were found (p<0.05) compared to control, as well as an higher intensity of all factors were found when compared to that of 21 day’s data, suggesting a treatment resistance. HET0016 inhibited tumor growth by reducing expression of different set of pro-angiogenic factors; however, a resistance to treatment seemed to happen after 21 days.

BMP-7 attenuates liver fibrosis via regulation of epidermal growth factor receptor.

Abstract: The aim of this study was to elucidate the effect of bone morphogenetic protein-7 (BMP-7) on liver fibrosis induced by carbon tetrachloride (CCl4) in vivo and on the hepatic stellate cells (HSC) activation in vitro. In vivo, thirty male ICR mice were randomly allocated to three groups, the control group (n = 6), the CCl4 group (n = 18) and the BMP-7+CCl4 group (n = 6). The model of liver fibrosis was induced by intraperitoneal injection with CCl4 three times per week lasting for 12 weeks in CCl4 group and the BMP-7+CCl4 group. After 8 weeks injection with CCl4, mice were intraperitoneal injected with human recombinant BMP-7 in BMP-7+CCl4 group. Meanwhile, mice in the CCl4 group were only intraperitoneal injection with equal amount of saline. The degree of liver fibrosis was assessed by HE and Masson's staining. PCR and western blot were used to detect mRNA and protein levels. In BMP-7+CCl4 group, serum levels of alanine aminotransferase (ALT) and aminotransferase (AST) were decreased and serum albumin (Alb) was increased. Meanwhile, the expressions of transforming growth factor-β1 (TGF-β1) and α-smooth muscle actin (α-SMA) were down-regulated by BMP-7 intervention as compared to the CCl4 group (P < 0.05). Furthermore, BMP-7 also suppressed the expression of epidermal growth factor receptor (EGFR) and phosphorylated-epidermal growth factor receptor (pEGFR). HE and Masson stain showed that liver damage was alleviated in BMP-7+CCl4 group. In vitro study, expression of EGFR, TGF-β1 and α-SMA were down regulated by BMP-7 dose-dependently, indicating it might effect on suppression of HSC activation. Therefore, our data indicate BMP-7 was capable of inhibiting liver fibrosis and suppressing HSCs activation, and these effects might rely on its crosstalk with EGFR and TGF-β1. We suggest that BMP-7 may be a potential reagentfor the prevention and treatment of liver fibrosis.

CB2 receptor activation ameliorates the proinflammatory activity in acute lung injury induced by paraquat

Abstract: Paraquat, a widely used herbicide, is well known to exhibit oxidative stress and lung injury. In the present study, we investigated the possible underlying mechanisms of cannabinoid receptor-2 (CB2) activation to ameliorate the proinflammatory activity induced by PQ in rats. JWH133, a CB2 agonist, was administered by intraperitoneal injection 1 h prior to PQ exposure. After PQ exposure for 4, 8, 24, and 72 h, the bronchoalveolar lavage fluid was collected to determine levels of TNF-α and IL-1β, and the arterial blood samples were collected for detection of PaO2 level. At 72 h after PQ exposure, lung tissues were collected to determine the lung wet-to-dry weight ratios, myeloperoxidase activity, lung histopathology, the protein expression level of CB2, MAPKs (ERK1/2, p38MAPK, and JNK1/2), and NF-κBp65. After rats were pretreated with JWH133, PQ-induced lung edema and lung histopathological changes were significantly attenuated. PQ-induced TNF-α and IL-1β secretion in BALF, increases of PaO2 in arterial blood, and MPO levels in the lung tissue were significantly reduced. JWH133 could efficiently activate CB2, while inhibiting MAPKs and NF-κB activation. The results suggested that activating CB2 receptor exerted protective activity against PQ-induced ALI, and it potentially contributed to the suppression of the activation of MAPKs and NF-κB pathways.