Immunohistochemical Staining For Caspase-3 Research Articles

Pelvic Radiation-Induced Testicular Damage: An Experimental Study at 1 Gray

ABSTRACTTherapeutic radiation of the pelvic region has been shown to cause damage to testicular germ cells. In this study we aimed to evaluate the effects of a low therapeutic dose of 1 Gy on the induction of cellular and histological damage in early-stage testicular germ cells and the impact of this radiation on offspring sex ratio. Unirradiated and irradiated male rats were mated with unirradiated female rats. Female rats were followed and the sex of the offspring was determined. The male rats were sacrificed at the end of the second week, and the testicular germ cells were subjected to genetic analysis along with cytological and histopathological examination. Sperm DNA was amplified with primers specific to testis-specific Y-linked protein, rat actin beta and testis-specific X-linked genes. The resulting products were separated by capillary electrophoresis. Histopathological changes were investigated by light microscopy along with the TUNEL assay and immunohistochemical staining for caspase-3. There was no significant difference between the two groups for sex ratio and size of offspring. The number of sperm cells bearing X or Y chromosomes’ did not differ significantly between these two groups. However, a 1 Gy dose of radiation caused significant cytopathological and histopathological changes in the testicular tissue. In the irradiated group, edematous regions were evident. The number of caspase-3 positive cells in the germinal epithelium of the seminiferous tubules was also significantly higher in the irradiated group. Our results showed that low-dose radiation induced apoptosis and caused significant cyto- and histopathological changes in the testicular tissue. Further research is required to fully elucidate their contribution to apoptosis and if low-dose radiation may potentially lead to long-term effects in the offspring. These results may also lead us to develop a new technique using the caspase-3 staining to monitor the susceptibility to low dose radiation.

Xerogenic potency of chronic usage of tramadol is related to structural changes in the parotid gland

Objectives: This study was designed to detect weather the xerogenic potency of chronic consumption of tramadol is related to its neural effect only or it is also related to structural changes of the parotid gland.Methods: 16 adult male albino rats weighing (200-250 gm) were divided into 3 groups; control group (4 rats) received distilled water, tramadol group (6 rats) received 40 mgkg of tramadol for 20 and 30 days, and tramadol withdrawal group (6 rats) received 40 mgkg of tramadol then tramadol was withdrawn for 20 and 30 days. Parotid gland was processed for HE after tramadol withdrawal the glands restored its normal architecture, mitotic and apoptotic activity were observed. Apoptotic findings were confirmed with the results of caspase-3 immuno-histochemical staining. Toluidine blue stain detected decreased amount of zymogen granules within the acinar cells of tramadol group which increased gradually after its withdrawal. Levels of MDA were increased in tramadol groups indicating increased level of oxidative stresses, after tramadol withdrawal MDA levels decreased again.Conclusion: Xerogenic potency of tramadol could be attributed to structural changes within the gland not only to its neural effect.

EVALUATION OF DOSE DEPENDENT EFFECTS OF GAMMA RADIATION ON RAT TONGUE INTERNAL STRUCTURES: HISTOMORPHOMETRIC AND IMMUNOHISTOCHEMICAL STUDY

Introduction: Radiotherapy is one of the common modalities in management of head and neck cancers. However, many injurious effects were reported on unavoidable oral tissues in the radiation field including the tongue and its heterogeneous internal structure. It is important to define the optimum dose of radiation with minimal hazards on the healthy tissues along with classification of the tissues according to their radio-sensitivity. Purpose: To evaluate histomorphometrically and immunohistochemically the dose dependent response of lingual internal structures of rats exposed to gamma radiation. Materials & Methods: Twenty-two adult male albino rats were divided into 3 groups: control group (6 rats), R1 group (8 rats) irradiated by 2.0 Gy whole-body gamma dose, and R2 group (8 rats) irradiated by 6.0 Gy. The effects on lingual internal structures were investigated after 3 days by histomorphometric and immunohistochemical staining for Caspase-3 using light microscopy. Results: There were a dose related changes in the minor salivary glands with more changes in serous glands. Mild changes were observed in muscles only at 6 Gy dose. The expression percentage of caspase-3 protein was significantly increased in a dose dependent manner in both of the irradiated groups when compared with control group P0.05. Conclusion: Gamma radiation especially 6 Gy dose adversely affects the internal structures of the tongue with the most affected are serous acini and the least are the muscle cells.

Investigation of the uterine structural changes in the experimental model with polycystic ovary syndrome and effects of vitamin D treatment: An ultrastructural and immunohistochemical study

In this study, we aimed to investigate the structural changes seen in the endometrium in experimental PCOS rat model and the effects of vitamin D treatment on these changes at immunohistochemical and electron microscopic levels. 24 prepubertal female rats were divided into three groups. Two groups were injected with dehydroepiandrosterone and one of them was treated with 1,25(OH)2 D3 at the same time. The control group was injected with sesame oil. At the end of the 28th day, the blood samples were collected. Uterus tissues were prepared for light and electron microscopic examinations. Epithelial, stromal and endometrial thickness measurements were investigated. Immunohistochemical staining was applied against caspase-3 and Ki-67. Serum AMH and estradiol levels were higher in PCOS group compared to the control group. Serum progesterone levels were similar in all groups. Endometrial, epithelial and stromal thickness measurements were increased in PCOS group compared to the control group, and decreased in the vitamin D treatment group compared to the PCOS group. Light and electron microscopic results of PCOS group showed an increase in apoptosis and proliferation. In the PCOS group, immunohistochemical staining of caspase-3 and Ki-67 were found to be higher than in the control group, but stainings were decreased with vitamin D treatment compared to PCOS group. Structural changes observed in endometrium may be related to implantation problems seen in patients with PCOS. Our studies suggest that vitamin D therapy may be beneficial in these patients.

The Ameliorating Effect of Sodium Selenite on the Histological Changes and Expression of Caspase-3 in the Testis of Monosodium Glutamate-Treated Rats: Light and Electron Microscopic Study.

Monosodium glutamate (MSG) is a commonly used flavor enhancer that may contribute to male infertility. Sodium selenite is inorganic chemical form of selenium (Se). Se is best known as an antioxidant. This study was designed to investigate the possible ameliorating role of sodium selenite against MSG-induced testicular toxicity and histological changes. Forty male albino rats were allocated into four groups. Control group received distilled water, SE group received sodium selenite (0.25 mg/kg/day) dissolved in distilled water orally, MSG group received MSG (6 mg/g/day) dissolved in distilled water orally, and MSG + SE group received both MSG and sodium selenite for 45 days. Testicular samples were prepared for biochemical, light, and electron microscopic studies. Immunohistochemical staining for caspase-3 was done. MSG group demonstrated a significant increase in malondialdehyde level, marked damage of seminiferous tubules with a significant reduction in diameter and height of the lining epithelium. Spermatogenic cells showed disorganization, dark nuclei, reduction in number, and maturation arrest. Vacuolations of interstitial tissue and Leydig cells were also observed. Percent area of fibrosis and caspase-3 immunoexpression was significantly increased. Ultrastructurally, irregular tubular basement membrane and damaged germ cells were found. The spermatogenic, Sertoli, and Leydig cells showed irregular shrunken nuclei, cytoplasmic vacuolations, and swollen mitochondria. MSG + SE group showed much better histological and ultrastructural picture and improvement of the measured biochemical and morphometric parameters. Percent area of caspase-3 immunoexpression was significantly decreased. In conclusion, sodium selenite ameliorated the testicular damaging effect of MSG through reduction of oxidative stress and apoptosis.

The protective effect of PFTα on alcohol-induced osteonecrosis of the femoral head.

Epidemiologic studies have shown alcohol plays a pivotal role in the development of osteonecrosis of the femoral head (ONFH). The aim of this study was to explore the underlying mechanism of alcohol-induced ONFH and the protective effect of pifithrin-α (PFTα). In vitro, we found ethanol treatment significantly activated p53, suppressed Wnt/β-catenin signaling and inhibited osteogenic-related proteins. Furthermore, by separating the cytoplasmic and nuclear proteins, we found ethanol inhibited osteogenesis by impairing the accumulation of β-catenin in both the cytoplasm and nucleus in human bone mesenchymal stem cells (hBMSCs), which resulted from activating glycogen synthase kinase-3β (GSK-3β). Therefore, PFTα, a p53 inhibitor, was introduced in this study to block the ethanol-triggered activation of p53 in hBMSCs and alcohol-induced ONFH in a rat model. In vivo, we established alcohol-induced ONFH in rats and investigated the protective effect of PFTα. Hematoxylin & eosin (H&E) staining combined with TdT-mediated dUTP nick end labeling (TUNEL), cleaved caspase-3 immunohistochemical staining, and micro-CT images revealed substantial ONFH in the alcohol-administered rats, whereas significantly less osteonecrosis developed in the rats injected with PFTα. Osteogenic-related proteins, including osteocalcin, osteopontin and collagen I, were significantly decreased in the alcohol-administered rats, whereas these results were reversed in the PFTα-injected rats. Fluorochrome labeling similarly showed that alcohol significantly reduced the osteogenic activity in the rat femoral head, which was blocked by the injection of PFTα. In conclusion, PFTα had an antagonistic effect against the effects of ethanol on hBMSCs and could be a clinical strategy to prevent the development of alcohol-induced ONFH.

Profile of bioactive compounds in Nymphaea alba L. leaves growing in Egypt: hepatoprotective, antioxidant and anti-inflammatory activity

BackgroundNymphaea alba L. represents an interesting field of study. Flowers have antioxidant and hepatoprotective effects, rhizomes constituents showed cytotoxic activity against liver cell carcinoma, while several Nymphaea species have been reported for their hepatoprotective effects. Leaves of N. alba have not been studied before. Therefore, in this study, in-depth characterization of the leaf phytoconstituents as well as its antioxidant and hepatoprotective activities have been performed where N. alba leaf extract was evaluated as a possible therapeutic alternative in hepatic disorders.MethodsThe aqueous ethanolic extract (AEE, 70%) was investigated for its polyphenolic content identified by high-resolution electrospray ionisation mass spectrometry (HRESI-MS/MS), while the petroleum ether fraction was saponified, and the lipid profile was analysed using gas liquid chromatography (GLC) analysis and compared with reference standards. The hepatoprotective activity of two doses of the extract (100 and 200 mg/kg; P.O.) for 5 days was evaluated against CCl4-induced hepatotoxicity in male Wistar albino rats, in comparison with silymarin. Liver function tests; aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), gamma glutamyl transpeptidase (GGT) and total bilirubin were performed. Oxidative stress parameters; malondialdehyde (MDA), reduced glutathione (GSH), catalase (CAT), superoxide dismutase (SOD), total antioxidant capacity (TAC) as well as inflammatory mediator; tumour necrosis factor (TNF)-α were detected in the liver homogenate. Histopathological examination of the liver and immunohistochemical staining of caspase-3 were performedResultsFifty-three compounds were tentatively identified for the first time in N.alba leaf extract, where ellagitannins represent the main identified constituents. Nine hydrocarbons, two sterols and eleven fatty acids were identified in the petroleum ether extract where, palmitic acid and linolenic acids represented the major saturated and unsaturated fatty acid respectively. N.alba AEE significantly improved the liver function, oxidative stress parameters as well as TNF-α in addition to the amelioration of histopathological features of the liver and a profound decrease in caspase-3 expression.ConclusionThese results shed light on the hepatoprotective effect of N. alba that is comparable with that of silymarin. The antioxidant activities of N. alba extract in addition to the inhibition of crucial inflammatory mediator, as TNF-α, might be the possible hepatoprotective mechanisms.

Antiseptic effect of sea cucumber (Holothuria atra) against multi-organ failure induced by sepsis: Molecular and histopathological study.

Sepsis is a systemic inflammatory response to infection and severe sepsis patients can develop acute lung and liver injury. The aim of the present study was to evaluate the efficacy of Holothuria atra methanolic body wall extract (HaE), as an antioxidant and anti-inflammatory agent against induced sepsis in a cecal ligation and puncture (CLP) rat model. In total, 30 males albino rats were divided into three groups (n=10 each) as follows: Sham (Sh), which was used as negative control; sepsis (Se), which was used as a positive control and was subjected to CLP surgery; and Ho, which was subjected to CLP and fed with 200 mg/kg (body weight) of HaE, once daily for 7 days. Subsequently, the expression of various genes was detected by polymerase chain reaction, while liver and lung tissues were examined by immunohistochemistry. The expression of Caspase-3 was significantly reduced in liver and lung tissues in the Ho group, while the expression levels of Gsta2, Cat and Sod1 genes were slightly reduced in the Ho group, when compared with the Se group. In addition, expression levels of tumor necrosis factor, interferon-γ, liver interleukin (IL)1b and lung IL1a were reduced in the Ho group compared with the Se group. Furthermore, histopathological changes were observed in liver tissues of the Se group, including congestion of hepatoportal blood vessel and focal hepatic necrosis, while lung tissues showed marked edema, hemorrhage and alveolar septal thickening. The Ho group showed apparent normal hepatic parenchyma and slight interstitial pneumonia. Immunohistochemical staining of caspase-3 in liver and lung tissues showed no expression in the Sh group, strong expression in the Se group and moderate expression in the Ho group. In conclusion, HaE demonstrated beneficial effect against induced sepsis, which may be attributed to its antioxidant and antiapoptotic activities.

The Effect of Intravitreal Azithromycin on the Albino Newborn Rabbit Retina.

Purpose: To evaluate the effect of intravitreal azithromycin on the retina in a newborn rabbit model.Methods: Twelve, two-week old New Zealand albino rabbits were divided into two groups (six in each). The right eyes of six rabbits received 0.75 mg (0.05 mL) azithromycin and the right eyes of the remaining six rabbits 1.5 mg (0.1 mL) azithromycin intravitreally. Left eyes were served as the control and received the same volume of saline. All eyes were enucleated at the third postinjection week. Retinal histology was examined by light microscopy. Apoptosis of the retinal cells was further evaluated by immunohistochemical staining for caspase-3 and in situ terminal deoxynucleotidyl transferase-mediated biotin-deoxyuridine triphosphate nick-end labeling (TUNEL) of DNA fragments. Results: Light microscopy demonstrated no retinal abnormalities in all eyes. However, retinal nuclear DNA fragmentation was evident in both study groups (33.6% with 1.5 mg and 21.4% with 0.75 mg azithromycin) with the TUNEL method. TUNEL staining ratio was statistically higher only in the second group treated with 1.5 mg azithromycin when compared to the control group (p=0.01 Mann Whitney U test). The ratio of caspase-3 positive cells in the two study groups was 21.5% and 20.2%, respectively. Caspase-3 staining ratio was statistically higher in both study groups when compared to the control eyes (p=0.00, p=0.00 respectively). The difference of TUNEL staining ratio between the two study groups was statistically significant (p=0.028), but there were no statistically significant differences in the two study groups by caspase-3 staining (p=0.247). Conclusion: In newborn rabbits, intravitreal azithromycin injection resulted in an apoptotic activity in the photoreceptor, bipolar and ganglion cells. Immunohistochemical analysis suggested that doses of 0.75 mg and 1.5 mg azithromycin, administered intravitreally might be toxic to the newborn rabbit retina.

Bioartificial liver attenuates intestinal mucosa injury and gut barrier dysfunction after major hepatectomy: Study in a porcine model

The aim of this study was to evaluate whether bioartificial liver support can attenuate gut mucosa injury in a porcine model of posthepatectomy liver dysfunction. Posthepatectomy liver failure was induced in pigs combining major (70%) liver resection and ischemia/reperfusion injury. An ischemic period of 150minutes was followed by reperfusion for 24hours. Animals were divided randomly into 2 groups: a control group (n=6) that received standard critical care and a bioartificial liver support group (Hepat, n=6) that were subjected to extracorporeal liver support for 6 hours during reperfusion. Intestinal mucosal injury, bacterial translocation, and endotoxin translocation were evaluated in all animals. Intestinal mucosa was also evaluated with markers of oxidative injury and immunohistochemical staining for caspase3. When compared with median values, the control group, animals in the Hepat group had a lesser intestinal mucosal injury score (4.0 [range:2.0-5.0] vs 1.0 [range:1.0-2.0]; P<.01), decreased bacterial translocation in the portal and the systemic circulation at 24hours of reperfusion (P<.05), and decreased portal and systemic endotoxin levels at 24hours (P<.05). At 24hours after reperfusion, mucosal protein carbonyls and malondialdehyde levels were decreased in Hepat animals (0.57nmol/mg [range:0.32-0.70] vs 0.33nmol/mg [range:0.03-0.53] and 3.85nmol/mg [range:3.01-6.43] vs 3.27nmol/mg [range:1.46-3.55], respectively; P<.05). There was no difference in tissue caspase staining. Bioartificial liver support seems to attenuate intestinal mucosal injury and gut barrier dysfunction after major hepatectomy.

Methylene Blue Protects against Toluene-Induced Brain Damage: Involvement of Nitric Oxide, NF-kB, and Caspase-3

The effect of methylene blue on brain oxidative stress and neuronal damage after toluene poisoning in rats was investigated. Rats were treated intraperitoneally with toluene at 900 mg/kg either alone or in combination with methylene blue at 5, 10, or 20 mg/kg daily for 6 days. The levels of malondialdehyde, nitrite, and reduced glutathione (GSH), and the activity of paraoxonase-1 (PON1) were evaluated in the brain and serum. We also measured serum butyrylcholinesterase (BChE) activity, brain derived neurorophic factor (BDNF), and nuclear factor kappa-B (NF-kB). Histopathological examination and immunohistochemical staining for caspase-3 and glial fibrillary acidic protein (GFAP) in the brain were performed. Toluene-treated rats exhibited increased lipid peroxidation (malondialdehyde) and nitrite levels in the brain and serum and reduced brain GSH level. There were also decreased PON1 and BChE activities, decreased BDNF level, and increased NF-kB level in the serum after toluene exposure. Neurodegeneration, cerebral edema, and marked degeneration of Purkinje cells were observed. There was increased caspase-3 immmunostaining, but decreased immmunostaining for GFAP in glial cells. Methylene blue induced a significant decrease in the levels of malondialdehyde and nitrite in the brain and serum of toluene-exposed rats. The GSH level did not change, but PON1 activity in the brain and serum increased. Methylene blue also decreased NF-kB level, increased BChE activity, and increased BDNF level in a dose-dependent manner. Methylene blue protected against the neuronal damaging effects of toluene, decreased caspase-3 immunoreactivity, and restored GFAP positivity. These findings indicate a neuroprotective effect for methylene blue against the brain damage induced by toluene. This protection is likely to involve the inhibition of nitric oxide, NF-kB, and caspase-3 by methylene blue treatment.

The effect of quercetin on cyclosporine-A-induced cardiotoxicity in adult male albino rats

Background Cyclosporine-A (Cs-A) is the most widely used immunosuppressive agent in organ transplantation and autoimmune disease treatment. Nevertheless, its therapeutic use is associated with serious effects, including cardiotoxicity. Quercetin is one of the naturally occurring flavonoids present in a lot of the human diets. Objective The aim of this study was to evaluate the effect of quercetin on cyclosporine-induced cardiotoxicity in adult male albino rats. Materials and methods Forty adult male rats were included and divided into four groups (10 rats each): group I was the control group; group II (the quercetin group) rats received daily intraperitoneal injection of quercetin (15 mg/kg body weight/day) for 4 weeks; group III (the cyclosporine group) rats received daily subcutaneous injection of Cs-A (20 mg/kg body weight/day) for 4 weeks; and group IV (the cyclosporine and quercetin group) rats received Cs-A and quercetin for 4 weeks. Heart specimens were taken and processed for light microscopy study (H&E and Masson’s trichrome staining and immunohistochemical staining for caspase-3) and electron microscopy study. Results Group III showed prominent myocardial fiber degeneration with areas of myofibril loss and interstitial hemorrhage. This was supported by the positive reaction for apoptotic marker caspase-3. An increase in the collagen fibers in the endomysium was obvious. Electron microscopic results showed irregular nucleus with disruption of the sarcolemma and intercalated discs. The cytoplasm showed an area of myofibril loss and mitochondrial swelling. Group IV showed improvement in most light and electron microscope changes described before. Conclusion Quercetin can be a natural cardioprotective agent and can be used to improve cyclosporine-induced cardiotoxicity, as it has antioxidant and anti-inflammatory enzyme activities. We advise taking a diet rich in quercetin or dietary supplementation.

Gelatin nanoparticle-mediated intranasal delivery of substance P protects against 6-hydroxydopamine-induced apoptosis: an in vitro and in vivo study.

BackgroundThe aim of this study was to investigate the protective role of intranasally administered substance P-loaded gelatin nanoparticles (SP-GNPs) against 6-hydroxydopamine (6-OHDA)-induced apoptosis in vitro and in vivo, and to provide a new strategy for treating brain pathology, such as Parkinson’s disease.MethodsSP-GNPs were prepared by a water-in-water emulsion method, and their stability, encapsulating efficiency, and loading capacity were evaluated. PC-12 cells were used to examine the enhancement of growth and inhibition of apoptosis by SP-GNPs in vitro using MTT assays. In the in vivo study, hemiparkinsonian rats were created by intracerebroventricular injection of 6-OHDA. The rats then received intranasal SP-GNPs daily for 2 weeks. Functional improvement was assessed by quantifying rotational behavior, and the degree of apoptosis was assessed by immunohistochemical staining for caspase-3 in the substantia nigra region.ResultsPC-12 cells with 6-OHDA-induced disease treated with SP-GNPs showed higher cell viability than their untreated counterparts, and cell viability increased as the concentration of substance P (SP) increased, indicating that SP could enhance cell growth and inhibit the cell apoptosis induced by 6-OHDA. Rats with 6-OHDA-induced hemiparkinsonism treated with SP-GNPs made fewer rotations and showed less staining for caspase-3 than their counterparts not treated with SP, indicating that SP protects rats with 6-OHDA-induced hemiparkinsonism from apoptosis and therefore demonstrates their functional improvement.ConclusionIntranasal delivery of SP-GNPs protects against 6-OHDA-induced apoptosis both in vitro and in vivo.

Investigation of protective role of curcumin against paclitaxel-induced inner ear damage in rats.

The aim of this study was to investigate the potential protective effect of curcumin on paclitaxel-induced ototoxicity in rats by means of immunohistochemical and histopathological analysis and distortion product otoacoustic emissions (DPOAEs). Animal study. Forty Sprague-Dawley rats were randomized into five groups. Group 1 was administered no paclitaxel and curcumin during the study. Groups 2, 3, 4 and 5 were administered 5 mg/kg paclitaxel; 200 mg/kg curcumin; 5 mg/kg paclitaxel, followed by 200 mg/kg curcumin; 200 mg/kg curcumin and a day later 5 mg/kg paclitaxel followed intraperitoneally by 200 mg/kg curcumin once a week for 4 consecutive weeks, respectively. After the final DPOAEs test, the animals were sacrificed and their cochlea were prepared for hematoxylin and eosin and caspase-3 staining. The DPOAEs thresholds and histopathological and immunohistochemical findings were substantially correlated in all groups. The histopathologic findings in the cochlea of the paclitaxel-treated animals showed not only changes in the organ of Corti, but also damage to the stria vascularis and spiral limbus, including nuclear degeneration, cytoplasmic vacuolization, and atrophy of intermediate cells. Additionally, cochlear changes in group 2, such as intense apoptosis, were confirmed by caspase-3 immunohistochemical staining. In group 4, coreceiving curcumin could not sufficiently prevent paclitaxel-induced ototoxicity, and the results in group 5 were similar to the control group. In our study, we have concluded that pre- and coreceiving curcumin can significantly protect the cochlear morphology and functions on paclitaxel-induced ototoxicity in rats. Curcumin might be considered as a potential natural product that, used as a dietary supplement, could be easily given to patients undergoing paclitaxel chemotherapy. NA

Protective effects of royal jelly against testicular damage in streptozotocin-induced diabetic rats

TO examine the effects of royal jelly (RJ) on testicular damage in streptozotocin (STZ)-induced diabetic rats. Eighteen adult Wistar albino rats were used, 6 in each of the 3 treatment groups: Group A: control, Group B: STZ-induced diabetes (untreated), Group C: STZ-induced diabetes plus RJ (400 mg/kg daily for 4 weeks). Diabetes was induced by a single intraperitoneal injection of STZ (60 mg/kg). Four weeks after the onset of diabetes, testicular apoptotic cell death was examined using immunohistochemical staining for caspase-3 and Ki67 staining for localization of proliferative cells. Compared with the control, the body and testicular weights of the RJ-treated and untreated diabetic rats were decreased (P < 0.05). The histopathological examination showed a significant increase in degenerative changes in the seminiferous tubules and in spermatogenesis of the STZ-treated rats. In contrast, the RJ treatment group showed near-normal morphology, in addition to an increased intensity of immunohistochemical staining for Ki67-positive cells. Diabetes induced a significant increase in testicular apoptotic cell death (caspase-3-positive cells). Caspase-3-positive cells were significantly decreased in the STZ plus RJ-treated group compared with the untreated STZ-induced diabetic group (P < 0.05).

A Multifunctional Polymeric Nanotheranostic System Delivers Doxorubicin and Imaging Agents across the Blood–Brain Barrier Targeting Brain Metastases of Breast Cancer

Metastatic brain cancers, in particular cancers with multiple lesions, are one of the most difficult malignancies to treat owing to their location and aggressiveness. Chemotherapy for brain metastases offers some hope. However, its efficacy is severely limited as most chemotherapeutic agents are incapable of crossing the blood-brain barrier (BBB) efficiently. Thus, a multifunctional nanotheranostic system based on poly(methacrylic acid)-polysorbate 80-grafted-starch was designed herein for the delivery of BBB-impermeable imaging and therapeutic agents to brain metastases of breast cancer. In vivo magnetic resonance imaging and confocal fluorescence microscopy were used to confirm extravasation of gadolinium and dye-loaded nanoparticles from intact brain microvessels in healthy mice. The targetability of doxorubicin (Dox)-loaded nanoparticles to intracranially established brain metastases of breast cancer was evaluated using whole body and ex vivo fluorescence imaging of the brain. Coexistence of nanoparticles and Dox in brain metastatic lesions was further confirmed by histological and microscopic examination of dissected brain tissue. Immuno-histochemical staining for caspase-3 and terminal-deoxynucleotidyl transferase dUTP nick end labeling for DNA fragmentation in tumor-bearing brain sections revealed that Dox-loaded nanoparticles selectively induced cancer cell apoptosis 24 h post-injection, while sparing normal brain cells from harm. Such effects were not observed in the mice treated with free Dox. Treatment with Dox-loaded nanoparticles significantly inhibited brain tumor growth compared to free Dox at the same dose as assessed by in vivo bioluminescence imaging of the brain metastases. These findings suggest that the multifunctional nanoparticles are promising for the treatment of brain metastases.

Does moxifloxacin alter oxidant status in the cornea? An experimental study

Objective: In this experimental study, we investigated the possible effects of intracameral moxifloxacin on oxidative stress parameters and endothelial cell morphology in corneal tissue.Methods: In total, 30 rats were randomly assigned to three groups of 10 rats: the sham group (Group 1, n = 10); the control group (Group 2), where balanced salt solution (BSS) was administered at a dose of 0.01 cc (n = 10); and the treatment group (Group 3), where moxifloxacin was administered at a dose of 0.05 mg/0.01 cc (n = 10). Total antioxidant status (TAS) and total oxidant status (TOS) in corneal tissue and blood samples were measured and the oxidative stress index (OSI) was calculated. Also, corneal tissue histopathology was evaluated with caspase-3 and caspase-8 staining. Apoptotic activity was also evaluated.Results: In blood samples, TAS, TOS, and OSI levels were not statistically significantly different (all p > 0.05). Compared with the sham and control groups, TOS and OSI levels in cornea tissue were significantly different in the moxifloxacin group (all p < 0.05). However, compared with the control group, no statistically significant difference was found in the sham group (all p > 0.05). Compared with the sham and control groups, apoptotic activity was higher in the moxifloxacin group, in both immunohistochemical staining for caspase-3 and caspase-8.Conclusions: Intracameral moxifloxacin injection seems to be safe systemically, but it may have toxic effects on corneal tissues, as suggested by oxidative stress parameters and a histopathological evaluation.

Detection of cell apoptosis in pelvic lymph nodes of patients with cervical cancer after neoadjuvant chemotherapy

To investigate if the administration of neoadjuvant chemotherapy (NACT) reduces pelvic lymph node metastasis by inducing tumour cell apoptosis in patients with cervical cancer. This study enrolled patients with stage Ib2-IIb cervical cancer who underwent surgery with (NACT group) or without (control group) prior cisplatin-based chemotherapy. Immunohistochemical staining of caspase-3 and an in situ terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling assay were used to measure the levels of apoptosis in primary tumours and pelvic lymph nodes. A total of 185 patients participated in the study: 102 in the NACT group and 83 in the control group. Treatment was considered to be clinically effective in 69.6% (71/102) of the NACT group. The rate of metastasis in the NACT group (20.6%; 21/102) was significantly lower than the control group (42.2%; 35/83). The level of caspase-3 immunostaining and the rate of apoptosis in primary tumours and pelvic lymph nodes in the NACT group were significantly higher than in the control group. NACT appeared to limit pelvic node metastasis by inducing tumour cell apoptosis in patients with cervical cancer.

Evaluation of the safety of intracameral trypan blue injection on corneal tissue using oxidative stress parameters and apoptotic activity: an experimental study.

The present experimental study aimed to investigate the effects of intracameral trypan blue (TB) on oxidative stress parameters and apoptosis in corneal tissue. Thirty rats were randomly assigned to three groups of 10 rats each: the sham group (Group 1); control group (Group 2); and treatment group (Group 3). The control group was administered 0.01 cc of balanced salt solution. The treatment group was administered 0.006 mg/0.01 cc of TB. The total antioxidant status (TAS) and total oxidant status (TOS) in corneal tissue and blood were measured and the oxidative stress index (OSI) was calculated. Finally, corneal tissue histopathology was evaluated using staining for caspase-3 and -8, and apoptotic activity was examined. The TAS, TOS and OSI levels in the blood samples were not significantly different (p>0.05 for all). Compared with the sham and control groups, the TOS and OSI levels in corneal tissue were significantly different in the treatment group (p<0.05 for all). No significant difference was observed between the sham group and the control group (p>0.05). Immunohistochemical staining for caspase-3 and caspase-8 demonstrated higher apoptotic activity in the TB group than in the sham and control groups. The present study showed that intracameral TB injection is safe systematically but may be toxic to corneal tissue, as demonstrated using oxidative stress parameters and histopathological evaluation.

Nox1 causes ileocolitis in mice deficient in glutathione peroxidase-1 and -2

We previously reported that mice deficient in two Se-dependent glutathione peroxidases, GPx1 and GPx2, have spontaneous ileocolitis. Disease severity depends on mouse genetic background. Whereas C57BL/6J (B6) GPx1/2-double-knockout (DKO) mice have moderate ileitis and mild colitis, 129S1Svlm/J (129) DKO mice have severe ileocolitis. Because GPx’s are antioxidant enzymes, we hypothesized that elevated reactive oxygen species trigger inflammation in these DKO mice. To test whether NADPH oxidase 1 (Nox1) contributes to colitis, we generated B6 triple-KO (TKO) mice to study their phenotype. Because the Nox1 gene is X-linked, we analyzed the effects of Nox1 on male B6 TKO mice and female B6 DKO mice with the Nox1+/− (het-TKO) genotype. We found that the male TKO and female het-TKO mice are virtually disease-free when monitored from 8 through 50 days of age. Male TKO and female het-TKO mice have nearly no signs of disease (e.g., lethargy and perianal alopecia) that are often exhibited in the DKO mice; further, the slower growth rate of DKO mice is almost completely eliminated in male TKO and female het-TKO mice. Male TKO and female het-TKO mice no longer have the shortened small intestine present in the DKO mice. Finally, the pathological characteristics of the DKO ileum, including the high level of crypt apoptosis (analyzed by apoptotic figures, TUNEL, and cleaved caspase-3 immunohistochemical staining), high numbers of Ki-67-positive crypt epithelium cells, and elevated levels of monocytes expressing myeloperoxidase, are all significantly decreased in male TKO mice. The attenuated ileal and colonic pathology is also evident in female het-DKO mice. Furthermore, the male DKO ileum has eightfold higher TNF cytokine levels than TKO ileum. Nox1 mRNA is highly elevated in both B6 and 129 DKO ileum compared to wild-type mouse ileum. Taking these results together, we propose that ileocolitis in the DKO mice is caused by Nox1, which is induced by TNF. The milder disease in female het-TKO intestine is probably due to random or imprinted X-chromosome inactivation, which produces mosaic Nox1 expression.