A histological,immunohistochemical and biochemical study of the effects of pomegranate peel extracts on gibberellic acid induced oxidative stress in adult rat testes

ABSTRACT

Gibberellins are commonly used plant growth regulators that exhibit deleterious effects on various animal tissues. We investigated the histological, immunohistochemical and biochemical effects of gibberellic acid (GA3) on rat testes as well as the possible protective role of pomegranate peel extract (PPE). We used 28 adult male rats divided into control, PPE treated, GS3 treated and GA3 + PPE treated groups. Testis specimens were analyzed for superoxide dismutase (SOD) and catalase (CAT) activity, and examined histologically. We also investigated the androgen receptor using immunohistochemistry. The GA3 treated group exhibited significantly decreased SOD and CAT levels and area percent of androgen receptor. Seminiferous tubules (ST) were widely separated and the germinal epithelium was separated from the basement membrane in some tubules. Areas of vacuolation, degenerated germ cells with pyknotic nuclei and large multinucleated cells were observed. Ultrastructurally, primary spermatocytes exhibited vacuolated cytoplasm, degenerated mitochondria and hyperchromatic nuclei. Degenerated early spermatids with a ruptured or hyperchromatic nucleus were found. Spermatozoa exhibited abnormalities of the head and tail. The cytoplasm of Sertoli and Leydig cells exhibited dilated smooth endoplasmic reticulum. A significant improvement of the biochemical, histological and immunohistochemical alterations was observed in the GA3 + PPE treated group compared to the GA3 treated group.     

Predictive markers of safety and immunogenicity of adjuvanted vaccines

Vaccination represents one of the greatest public health triumphs; in part due to the effect of adjuvants that have been included in vaccine preparations to boost the immune responses through different mechanisms. Although a variety of novel adjuvants have been under development, only a limited number have been approved by regulatory authorities for human vaccines. This report reflects the conclusions of a group of scientists from academia, regulatory agencies and industry who attended a conference on the current state of the art in the adjuvant field. Held at the U.S. Pharmacopeial Convention (USP) in Rockville, Maryland, USA, from 18 to 19 April 2013 and organized by the International Association for Biologicals (IABS), the conference focused particularly on the future development of effective adjuvants and adjuvanted vaccines and on overcoming major hurdles, such as safety and immunogenicity assessment, as well as regulatory scrutiny. More information on the conference output can be found on the IABS website, http://www.iabs.org/.     

Ecosystem Services from Keystone Species: Diversionary Seeding and Seed‐Caching Desert Rodents Can Enhance Indian Ricegrass Seedling Establishment

Seeds of Indian ricegrass (Achnatherum hymenoides), a native bunchgrass common to sandy soils on arid western rangelands, are naturally dispersed by seed‐caching rodent species, particularly Dipodomys spp. (kangaroo rats). These animals cache large quantities of seeds when mature seeds are available on or beneath plants and recover most of their caches for consumption during the remainder of the year. Unrecovered seeds in caches account for the vast majority of Indian ricegrass seedling recruitment. We applied three different densities of white millet (Panicum miliaceum) seeds as “diversionary foods” to plots at three Great Basin study sites in an attempt to reduce rodents' over‐winter cache recovery so that more Indian ricegrass seeds would remain in soil seedbanks and potentially establish new seedlings. One year after diversionary seed application, a moderate level of Indian ricegrass seedling recruitment occurred at two of our study sites in western Nevada, although there was no recruitment at the third site in eastern California. At both Nevada sites, the number of Indian ricegrass seedlings sampled along transects was significantly greater on all plots treated with diversionary seeds than on non‐seeded control plots. However, the density of diversionary seeds applied to plots had a marginally non‐significant effect on seedling recruitment, and it was not correlated with recruitment patterns among plots. Results suggest that application of a diversionary seed type that is preferred by seed‐caching rodents provides a promising passive restoration strategy for target plant species that are dispersed by these rodents.     

Oxidative stress: Determination of 4-hydroxy-2-nonenal by gas chromatography/mass spectrometry in human and rat plasma

Abstract

The lipid peroxidation product 4-hydroxynonenal (HNE) is a biomarker of oxidative stress which is essentially involved in the pathophysiology of many diseases. The analysis of HNE is challenging because this aldehyde is extremely reactive and thus unstable. Hence, we adopted a gas chromatography–mass spectrometry (GC–MS) method based on derivatization of HNE with pentafluorobenzylhydroxylamine–HCl followed by trimethylsilylation to trimethylsilyl ethers. Ions representative for a negative ion chemical ionization mode were recorded at m/z = 152 for HNE and at m/z = 162 for the deuterated analogon (HNE-d₁₁) as internal standard. This excellent stable and precise GC–MS method was carefully validated for HNE, and showed good linearity (r² = 0.998), and high specificity and sensitivity. Within-day precisions were 4.4–6.1% and between-day precisions were 5.2–10.2%. Accuracies were between 99% and 104% for the whole calibration range (2.5–250 nmol/L) of HNE.

To examine the versatility of this modified GC–MS method, we analyzed HNE in ethylenediaminetetraacetic acid (EDTA) plasma in a well-defined collective of migraine patients; recently published. The results underline our former observations that women with migraine are afflicted with increased levels of HNE. Patients with thyroidal dysfunction showed no significant HNE alterations. This was confirmed by normal HNE EDTA plasma levels in hyper- und hypothyroid Sprague-Dawley rats.

Taken together, the GC–MS method presented herein is of excellent quality to record oxidative stress-related bioactive HNE levels. This is important for a reorientation of oxidative stress analytics in other human diseases first of atherosclerosis and cancer.     

Relationship between serum reactive oxidative metabolite level and skin reaction in an irradiated rat model

Abstract

Purpose. Ionizing radiation generates free radicals and reactive oxygen species that induce DNA damage in vivo. This study aimed to determine the relationship between serum reactive oxygen metabolite (ROM) levels and skin reaction after irradiation in a rat model. Methods and materials. I. Female Wistar rats were classified into 0 Gy (control), 2 Gy, and 30 Gy groups; serum ROM levels were measured in the very acute phase. II. Other female Wistar rats were classified into 0 Gy (control), 30 Gy, 50 Gy, and 70 Gy groups; serum ROM levels were measured before and 3, 7, 16, 24, 31, and 38 days after irradiation. Skin reaction was evaluated according to the SRS (0–5) twice every week. Results. Serum ROM levels in the subacute phase were significantly higher in the 50 and 70 Gy groups than in the 0 and 30 Gy groups [p = 0.029, repeated-measure analysis of variance (ANOVA)]. As expected, SRSs increased in the order of the 0 Gy, 30 Gy, 50 Gy, and 70 Gy groups and differed significantly among these groups (p < 0.001, repeated-measure ANOVA). Peak serum ROM levels were observed 16 days after irradiation in all irradiated groups and corresponded with the appearance of visible skin reaction after irradiation. Conclusions. Serum ROM levels may be useful for evaluating radiation damage in mammals. Further investigations are required to investigate changes in intracellular metabolism after irradiation at gene and protein levels.     

A New Coronary Retroinfusion Technique in theRat Infarct Model: Transjugular Cardiac Vein Catheterization

Cell delivery via the retrograde coronary route boasts less vessel embolism, myocardial injury, and arrhythmogenicity when compared with those via antegrade coronary administration or myocardial injection. However, conventional insertion into the coronary sinus and consequent bleeding complication prevent its application in small animals. To overcome the complication of bleeding, we described a modified coronary retroinfusion technique via the jugular vein route in rats with myocardial infarction (MI). A flexible wire with a bent end was inserted into the left internal jugular vein and advanced slowly along the left superior vena cava. Under direct vision, the wire was run into the left cardiac vein by rotating the wire and changing the position of its tip. A fine tube was then advanced along the wire to the left cardiac vein. This modified technique showed less lethal hemorrhage than the conventional technique. Retroinfusion via transjugular catheter enabled efficient fluid or cell dissemination to the majority areas of the free wall of the left ventricle, covering the infarcted anterior wall. In conclusion, transjugular cardiac vein catheterization may make retrocoronary infusion a more safe and practical route for delivering cell, drug, and gene therapy into the infarcted myocardium of rats.     

A High-throughput Method for Measurement of Glomerular Filtration Rate in Conscious Mice

The measurement of glomerular filtration rate (GFR) is the gold standard in kidney function assessment. Currently, investigators determine GFR by measuring the level of the endogenous biomarker creatinine or exogenously applied radioactive labeled inulin (³H or ¹⁴C). Creatinine has the substantial drawback that proximal tubular secretion accounts for ~50% of total renal creatinine excretion and therefore creatinine is not a reliable GFR marker. Depending on the experiment performed, inulin clearance can be determined by an intravenous single bolus injection or continuous infusion (intravenous or osmotic minipump). Both approaches require the collection of plasma or plasma and urine, respectively. Other drawbacks of radioactive labeled inulin include usage of isotopes, time consuming surgical preparation of the animals, and the requirement of a terminal experiment. Here we describe a method which uses a single bolus injection of fluorescein isothiocyanate-(FITC) labeled inulin and the measurement of its fluorescence in 1-2 μl of diluted plasma. By applying a two-compartment model, with 8 blood collections per mouse, it is possible to measure GFR in up to 24 mice per day using a special work-flow protocol. This method only requires brief isoflurane anesthesia with all the blood samples being collected in a non-restrained and awake mouse. Another advantage is that it is possible to follow mice over a period of several months and treatments (i.e. doing paired experiments with dietary changes or drug applications). We hope that this technique of measuring GFR is useful to other investigators studying mouse kidney function and will replace less accurate methods of estimating kidney function, such as plasma creatinine and blood urea nitrogen.     

Analytical FcRn affinity chromatography for functional characterization of monoclonal antibodies

The neonatal Fc receptor (FcRn) is important for the metabolic fate of IgG antibodies in vivo. Analysis of the interaction between FcRn and IgG in vitro might provide insight into the structural and functional integrity of therapeutic IgG that may affect pharmacokinetics (PK) in vivo. We developed a standardized pH gradient FcRn affinity liquid chromatography method with conditions closely resembling the physiological mechanism of interaction between IgG and FcRn. This method allows the separation of molecular IgG isoforms, degradation products and engineered molecules based on their affinity to FcRn. Human FcRn was immobilized on the column and a linear pH gradient from pH 5.5 to 8.8 was applied. FcRn chromatography was used in comparison to surface plasmon resonance to characterize different monoclonal IgG preparations, e.g., oxidized or aggregated species. Wild-type and engineered IgGs were compared in vitro by FcRn chromatography and in vivo by PK studies in huFcRn transgenic mice. Analytical FcRn chromatography allows differentiation of IgG samples and variants by peak pattern and retention time profile. The method can distinguish: 1) IgGs with different Fabs, 2) oxidized from native IgG, 3) aggregates from monomer and 4) antibodies with mutations in the Fc part from wild-type IgGs. Changes in the FcRn chromatographic behavior of mutant IgGs relative to the wild-type IgG correlate to changes in the PK profile in the FcRn transgenic mice. These results demonstrate that FcRn affinity chromatography is a useful new method for the assessment of IgG integrity.     

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.