Size- and Age-Dependent Neurotoxicity of Engineered Metal Nanoparticles in Rats

Earlier we showed that chronic administration of engineered nanoparticles (NPs) from metals, e.g., Cu, Ag, or Al (50–60 nm, 50 mg/kg, i.p. daily for 1 week) alter blood–brain barrier (BBB) disruption and induce brain pathology in adult rats (age 18 to 22 weeks). However, effects of size-dependent neurotoxicity of NPs in vivo are still largely unknown. In present investigation, we examined the effects of different size ranges of the above-engineered NPs on brain pathology in rats. Furthermore, the fact that age is also an important factor in brain pathology was also investigated in our rat model. Our results showed that small-sized NPs induced the most pronounced BBB breakdown (EBA +480 to 680 %; radioiodine +850 to 1025 %), brain edema formation (+4 to 6 %) and neuronal injuries (+30 to 40 %), glial fibrillary acidic protein upregulation (+40 to 56 % increase), and myelin vesiculation (+30 to 35 % damage) in young animals as compared to controls. Interestingly, the oldest animals (30 to 35 weeks of age) also showed massive brain pathology as compared to young adults (18 to 20 weeks old). The Ag and Cu exhibited greater brain damage compared with Al NPs in all age groups regardless of their size. This suggests that apart from the size, the composition of NPs is also important in neurotoxicity. The very young and elderly age groups exhibited greater neurotoxicity to NPs suggests that children and elderly are more vulnerable to NPs-induced brain damage. The NPs-induced brain damage correlated well with the upregulation of neuronal nitric oxide synthase activity in the brain indicating that NPs-induced neurotoxicity may be mediated via increased production of nitric oxide, not reported earlier.     

Neuroprotective Effect of Chitosan Oligosaccharide on Hypoxic-Ischemic Brain Damage in Neonatal Rats

Neonatal hypoxic-ischemic brain damage (HIBD) is one of the leading causes of neonatal mortality and permanent neurological disability worldwide and the effective treatment strategies are not yet available. It has been demonstrated that Chitosan oligosaccharide (COS) exerts protective effect in vitro ischemic brain injury. However, no information is available on possible effects of COS on neonatal HIBD. To investigate the hypothesis of the potential neuroprotective effect of COS on the brain injury due to HIBD, 7-day-old Sprague–Dawley rats were treated with left carotid artery ligation followed by exposure to 8% oxygen (balanced with nitrogen) for 2.5 h at 37?°C. After COS treatment, the cerebral damage was measured by behavior tasks, 2,3,5-triphenyltetrazolium chloride(TTC), Hematoxyline-Eosin(HE), Nissl and Fluoro-Jade B(FJB)staining. In addition, the oxidative stress were assayed with ipsilateral hemisphere homogenates. Immunofluorescence staining were used to examine the activation of the astrocyte and microglia. Expression of inflammatory-related proteins were analyzed by western-blot analysis. In this study we found that administration of COS ameliorated early neurological reflex behavior, significantly reduce brain infarct volume and attenuated neuronal cell injury and degeneration. Furthermore, COS markedly decreased the level of MDA, lactic acid and increased SOD, GSH-Px and T-AOC. COS attenuated hypoxic-ischemic induced up-regulation of expressions of interleukin-1β (IL-1β), tumor necrosis factor alpha (TNF-α), meanwhile it dramatically increased the interleukin-10 (IL-10). These results suggest that COS exerts neuroprotection on hypoxic-ischemic brain damage in neonatal rats, it implies COS might be a potential therapeutic for the treatment of HIBD.     

Neurotoxicity of Glucocorticoids in the Primate Brain

Severe and prolonged physical and psychological stress is known to cause brain damage; long-term torture victims in prison bare later developed psychiatric disorders and cerebral cortical atrophy observed in CT scans (Jensen, Genefke, Hyldebrandt, Pedersen, Petersen, and Weile. 1982). In nonhuman primates, we observed degeneration and depletion of the hippocampal neurons in African green monkeys that had been severely abused by cagemates and died with complications of multiple gastric ulcers and adrenal cortical hyperplasia (Uno, Tarara, Else, Suleman and Sapolsky, 1989). In our previous studies the administration of dexamethasone (DEX) (5 mg/kg) to pregnant rhesus monkeys at 132 to 133 days of gestation induced degeneration and depletion of the hippocampal pyramidal and dentate granular neurons in the brains of 135-gestation-day fetuses, and these changes were retained in the brains of fetuses at near term, 165 days of gestation (Uno, Lohmiller, Thieme, Kemnitz, Engle, Roecker, and Farrell, 1990). We also found that implantation of a cortisol pellet in the vicinity of the hippocampus in adult vervet monkeys induced degeneration of the CA3 pyramidal neurons and their dendritic branches (Sapolsky, Uno, Rebert, and Finch, 1990). Thus, hippocampal pyramidal neurons containing a high concentration of glucocorticoid receptors appear to be highly vulnerable to either hypercortisolemia caused by severe stress or to exposure to exogenous glucocorticoids. To study the long-term postnatal sequelae of prenatal brain damage, eight rhesus monkeys were treated with either DEX (5 mg/kg), 5 animals, or vehicle, 3 animals, at 132 to 133 days of gestation. After natural birth, all animals lived with their mothers for 1 year. At 9 months of age, we found that DEX-treated animals had significantly high plasma cortisol at both base and post stress (isolation) levels compared to age-matched vehicle-treated animals. Magnetic resonance images (MRI) of the brain at 20 months of age showed an approximately 30% reduction in size and segmental volumes of the hippocampus in DEX-treated compared to vehicle-treated animals. Measurements of whole brain volume by MRI showed no significant differences between DEX and vehicle groups. Prenatal administration of a potent glucocorticoid (DEX) induced an irreversible deficiency of the hippocampal neurons and high plasma cortisol at the circadian baseline and post-stress levels in juvenile rhesus monkeys. These results suggest that the hippocampus mediates negative feedback of cortisol release; a lack or deficiency of the hippocampal neurons attenuates this feedback resulting in hypercortisolemia. The hippocampal deficiency in rhesus monkeys induced by prenatal administration of DEX appears to be a good model for neuroendocrinological dysfunctions and hippocampal development in human juveniles whose mothers were exposed to severe stress or received a high dose of glucocorticosteroids during pregnancy.     

Systemic Evaluation of Hypoxic-Ischemic Brain Injury in Neonatal Rats

The objective of the study was to evaluate the systematically rat model of neonatal hypoxic-ischemic brain damage. The right carotid arteries of 7-day-old healthy Wistar rats were ligated, and then, the rats were subjected to an environment with 8 % of oxygen. Four weeks after the birth, neurobehavioral test, water maze test, and motor-evoked potential and neuropathologic examinations were performed. The footprint analysis showed significantly larger and instable paces in the hypoxic-ischemic group (P < 0.05); the time that rats crossed the balance beam in the hypoxic-ischemic group was longer than the control group (P < 0.05). The water maze test showed that the escape latency of hypoxic-ischemic group was significantly longer than that of control group (P < 0.05). The hindlimb quadriceps compound muscle-evoked potential CMEP of rats in hypoxic-ischemic group showed that the wave amplitude was lower than that of control group (P < 0.05). HE staining showed visible periventricular leukomalacia in hypoxic-ischemic groups; disrupted nuclear membrane was detected in the IH group with transelectronmicroscopy; Immunohistochemistry: compared with control group, MBP-positive neurocytes decreased, glial fibrillary acidic protein positive neurocytes increased in the periventricular zone (P < 0.05). Carotid artery ligation combining the hypoxic chamber created a reliable and stable rat model of neonatal hypoxic-ischemic brain damage and can be used for experimental research related to management of cerebral palsy.     

Effect of Polysorbate 80 Quality on Photostability of a Monoclonal Antibody

Polysorbate 80 is one of the key components of protein formulations. It primarily inhibits interfacial damage of the protein molecule due to mechanical stress during shipping and handling. However, polysorbate 80 also affects the formulation photostability. Exposure to light of polysorbate 80 aqueous solution results in peroxide generation, which in turn may result in oxidation of the susceptible amino acid residues in the protein molecule. The purpose of this study was to determine if the photostability of our proprietary IgG(1) monoclonal antibody formulation containing polysorbate 80 is affected by the quality (grade/vendor) of polysorbate 80. Following four types of polysorbate 80 were tested: (1) Polysorbate 80 Super-Refined, Mallinckrodt Baker, (2) Polysorbate 80 NF, Mallinckrodt Baker, (3) Polysorbate 80 NF, EMD Chemicals, and (4) Ultra-pure Polysorbate 80 (HX), NOF Corporation. The samples were exposed to light as per ICH guidelines Q1B. The results of the study show that photostability of the antibody formulation is indeed affected by the quality of polysorbate 80. This study underscores the importance of carefully choosing the quality of polysorbate 80 to ensure the robustness of formulation.     

Changes in Small Intestinal Morphology and Digestive Enzyme Activity with Oral Administration of Copper-Loaded Chitosan Nanoparticles in Rats

The experiment was conducted to evaluate the effect of copper-loaded chitosan nanoparticles on the small intestinal morphology and activities of digestive enzyme and mucosal disaccharase in rats. Forty male Sprague–Dawley rats, with average body weight of 82 g, were randomly allotted to five groups (n = 8). All rats were received a basal diet (control) or the same basal diet added with 80 mg/kg BW CuSO₄, 80 mg/kg BW chitosan (CS-I), 80 mg/kg BW copper-loaded chitosan nanoparticles (CSN-I), 160 mg/kg BW copper-loaded chitosan nanoparticles (CSN-II), respectively. The experiment lasted 21 days. The results showed that the villus heights of the small intestinal mucosa in groups CSN-I and CSN-II were higher than those of the control, group CuSO₄ or CS-I. The crypt depth of duodenum and ileum mucosa in group CSN-I or CSN-II was depressed. Compared with the control, there were no significant effects of CuSO₄ or CS-I on the villus height and crypt depth of small intestinal mucosa. Supplementation with CSN improved the activities of trypsin, amylase and lipase in the small intestinal contents and maltase, sucrase and lactase of duodenum, jejunum, and ileum mucosa while there were no significant effects of CuSO₄ on the digestive enzyme activities of the small content compared with the control. The results indicated that intestinal morphology, activities of digestive enzyme in digesta and mucosal disaccharase were beneficially changed by treatment of copper-loaded chitosan nanoparticles.     

Biocidal Activity of Chitosan Aspartate Nanoparticles

Microbiology - The biological activity of chitosan aspartate particles formed in the chitosan‒L-aspartic acid‒water system at the initial stage of phase separation was investigated....     

Effect of Polysorbate 80 Concentration on Thermal and Photostability of a Monoclonal Antibody

Polysorbate 80 is widely used in protein formulations to protect protein against agitation-induced aggregation. In this study, we address concerns about residual peroxide present in Polysorbate 80 on protein stability. Residual peroxide may oxidize active pharmaceutical ingredients leading to reduced stability and may ultimately lead to lower potency and efficacy. The effect of Polysorbate 80 concentration on thermal and photostability of monoclonal antibody of the IgG1 subclass (MAb1) was evaluated at Polysorbate 80 concentrations ranging from 0.00% to 1.00% (w/v). MAb1 samples at 5 mg/mL with various Polysorbate 80 concentrations were subjected to accelerated thermal stress by incubation at 25°C, 40°C, and 50°C for a period of 4 weeks and light stress per ICH guideline Q1B, option 1. Our results show that Polysorbate 80 concentration of 1.00% (w/v) adversely affected thermal and photostability of MAb1. This study demonstrates the importance of carefully choosing Polysorbate 80 concentration in protein formulations to prevent destabilizing effect of Polysorbate 80 on thermal and photostability.     

Tissue Distribution and Immunocytochemical Localization of Neurotrophin-3 in the Brain and Peripheral Tissues of Rats

Abstract: The tissue distribution of neurotrophin-3 (NT-3) was investigated in rats at 1 month of age using a newly established, sensitive two-site enzyme immunoassay system for NT-3, as well as the immunocytochemical localization of this protein. The immunoassay for NT-3 enabled us to quantify NT-3 at levels > 3 pg per assay. In the rat brain, NT-3 was detectable only in the olfactory bulb (0.54 ng/g wet weight), cerebellum (0.71 ng/g), septum (0.91 ng/g), and hippocampus (6.3 ng/g). By contrast, NT-3 was widely distributed in peripheral tissues. Appreciable levels of NT-3 were also found in the thymus (31 ng/g), heart (38 ng/g), diaphragm (21 ng/g), liver (45 ng/g), pancreas (892 ng/g), spleen (133 ng/g), kidney (40 ng/g), and adrenal gland (46 ng/g). An antibody specific for NT-3 bound to pyramidal cells in the CA2-CA4 regions of the hippocampus, to A cells in the islets of Langerhans in the pancreas, to unidentified cells in the red pulp of the spleen, to liver cells, and to muscle fibers in the diaphragm from rats at 1 month of age. Molecular masses of NT-3-immunoreactive proteins in the hippocampus and pancreas were 14 and 12 kDa, respectively. Thus, in rats, NT-3 was detected in restricted regions of the brain and in the visceral targets of the nodose ganglia at high concentrations. Our present results suggest that NT-3 not only functions as a classical target-derived neurotrophic factor but also can play other roles.     

Development of Timolol-Loaded Galactosylated Chitosan Nanoparticles and Evaluation of Their Potential for Ocular Drug Delivery

This study was conducted to develop timolol maleate (TM)-loaded galactosylated chitosan (GC) nanoparticles (NPs) (TM-GC-NPs) followed by optimization via a four-level and three-factor Box–Behnken statistical experimental design. The optimized nanoparticles showed a particle size of 213.3?±?6.83 nm with entrapment efficiency of 38.58?±?1.31% and drug loading of 17.72?±?0.28%. The NPs were characterized with respect to zeta potential, pH, surface morphology, and differential scanning calorimetry (DSC). The determination of the oil–water partition coefficient demonstrated that the TM-GC-NPs had a high liposolubility at pH 6 as compared to timolol-loaded chitosan nanoparticles (TM-CS-NPs) and commercial TM eye drops. The in vitro release study indicated that TM-GC-NPs had a sustained release effect compared with the commercial TM eye drops. Ocular tolerance was studied by the hen’s egg chorioallantoic membrane (HET-CAM) assay and the formulation was non-irritant and could be used for ophthalmic drug delivery. The in vitro transcorneal permeation study and confocal microscopy showed enhanced penetration, and retention in the cornea was achieved with TM-GC-NPs compared with the TM-CS-NPs and TM eye drops. Preocular retention study indicated that the retention of TM-GC-NPs was significantly longer than that of TM eye drops. The in vivo pharmacodynamic study suggested TM-GC-NPs had a better intraocular pressure (IOP) lowering efficacy and a prolonged working time compared to commercial TM eye drops (P?≤?0.05). The optimized TM-GC-NPs could be prepared successfully promising their use as an ocular delivery system.     

Serratiopeptidase Loaded Chitosan Nanoparticles by Polyelectrolyte Complexation: In Vitro and In Vivo Evaluation

The aim of the present study was to formulate serratiopeptidase (SER)-loaded chitosan (CS) nanoparticles for oral delivery. SER is a proteolytic enzyme which is very sensitive to change in temperature and pH. SER-loaded CS nanoparticles were fabricated by ionic gelation method using tripolyphosphate (TPP). Nanoparticles were characterized for its particle size, morphology, entrapment efficiency, loading efficiency, percent recovery, and in vitro dissolution study. SER-CS nanoparticles had a particle size in the range of 400–600 nm with polydispersity index below 0.5. SER association was up to 80 ± 4.2%. SER loading and CS/TPP mass ratio were the primary parameters having direct influence on SER-CS nanoparticles. SER-CS nanoparticles were freeze dried using trehalose (20%) as a cryoprotectant. In vitro dissolution showed initial burst followed by sustained release up to 24 h. In vivo anti-inflammatory activity was carried out in rat paw edema model. In vivo anti-inflammatory activity in rat paw edema showed prolonged anti-inflammatory effect up to 32 h relative to plain SER.KEY WORDS: anti-inflammatory activity, chitosan, nanoparticle, serratiopeptidase, TPP     

Intranasal Drug Delivery of Olanzapine-Loaded Chitosan Nanoparticles

The aim of this study was to investigate olanzapine (OZ) systemic absolute bioavailability after intranasal (i.n.) administration in vivo to conscious rabbits. Furthermore, the study investigated the potential use of chitosan nanoparticles as a delivery system to enhance the systemic bioavailability of olanzapine following intranasal administration. Olanzapine-loaded chitosan nanoparticles were prepared through ionotropic gelation of chitosan with tripolyphosphate anions and studied in terms of their size, drug loading, and in vitro release. The OZ nanoparticles were administered i.n. to rabbits, and OZ plasma concentration at predetermined time points was compared to i.n. administration of OZ in solution. The concentrations of OZ in plasma were analyzed by ultra performance liquid chromatography mass spectroscopy (UPLC/MS). OZ-loaded chitosan nanoparticles significantly (p < 0.05) enhanced systemic absorption with 51 ± 11.2% absolute bioavailability as compared to 28 ± 6.7% after i.n. administration of OZ solution. The results of the present study suggest that intranasal administration of OZ-loaded chitosan nanoparticles formulation could be an attractive modality for delivery of OZ systemically.KEY WORDS: bioavailability, intranasal, nanoparticles, olanzapine, pharmacokinetic     

Sodium Nitrite Alone Protects the Brain Microsomal Ca2+-ATPase Against Potassium Cyanide-induced Neurotoxicity In Rats

The effect of a short-term oral administration of potassium cyanide (KCN) (200 ppm in diet) with or without sodium nitrite (NaNO₂) pretreatment on rat brain microsomal Ca^2± ATPase was investigated. The specific activity value of the enzyme significantly decreased (p<0.05) by 50% compared with control and by 63% for KCN-treated rats compared with KCN-treated rats pretreated with NaNO₂. There was no significant difference at the h=0.05 level between the values obtained for the control and KCN-treated rats pretreated with NaNO₂. These results show both that feeding lowers brain microsomal Ca²⁺-ATPase activity and that NaNO₂ has a protective role (antidote function) in that respect.     

Obtaining of Metal Nanoparticles Using Reducing Agents and Chitosan

Applied Biochemistry and Microbiology - Unique features of metal nanoparticles (NPs) are attracting the attention of researchers from various fields of science, including biomedicine, microbiology,...     

Juxtacellular Monitoring and Localization of Single Neurons within Sub-cortical Brain Structures of Alert,Head-restrained Rats

There are a variety of techniques to monitor extracellular activity of single neuronal units. However, monitoring this activity from deep brain structures in behaving animals remains a technical challenge, especially if the structures must be targeted stereotaxically. This protocol describes convenient surgical and electrophysiological techniques that maintain the animal’s head in the stereotaxic plane and unambiguously isolate the spiking activity of single neurons. The protocol combines head restraint of alert rodents, juxtacellular monitoring with micropipette electrodes, and iontophoretic dye injection to identify the neuron location in post-hoc histology. While each of these techniques is in itself well-established, the protocol focuses on the specifics of their combined use in a single experiment. These neurophysiological and neuroanatomical techniques are combined with behavioral monitoring. In the present example, the combined techniques are used to determine how self-generated vibrissa movements are encoded in the activity of neurons within the somatosensory thalamus. More generally, it is straightforward to adapt this protocol to monitor neuronal activity in conjunction with a variety of behavioral tasks in rats, mice, and other animals. Critically, the combination of these methods allows the experimenter to directly relate anatomically-identified neurophysiological signals to behavior.     

Effects of Flaxseed Oil on Lead Acetate-Induced Neurotoxicity in Rats

It is well known that chronic exposure to lead (Pb(+2)) alters a variety of behavioral tasks in rats and mice. Here, we investigated the effect of flaxseed oil (1,000?mg/kg) on lead acetate (20?mg/kg)-induced brain oxidative stress and neurotoxicity in rats. The levels of Pb(+2), lipid peroxidation, nitric oxide (NO), and reduced glutathione (GSH) and the activity of catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GR), glutathione-S-transferase (GST), and glutathione peroxidase (GPx) were determined in adult male albino rats. The level of Pb(+2) was markedly elevated in brain and blood of rats. This leads to enhancement of lipid peroxidation and NO production in brain with concomitant reduction in GSH, CAT, SOD, GR, GST, and GPx activities. These findings were associated with DNA fragmentation. In addition, lead acetate induced brain injury as indicated by histopathological changes of the brain. Treatment of rats with flaxseed oil resulted in marked improvement in most of the studied parameters as well as histopathological features. These findings suggest to the conclusion that flaxseed oil significantly decreased the adverse harmful effects of lead acetate exposure on the brain as well as Pb(+2)-induced oxidative stress.     

Localization of Glycogen Synthase in Brain

Antisera against glycogen synthase from canine brain were prepared and used for investigation of the localization of the enzyme in the brain. Antisera cross-reacted only with the 88-kilodalton protein that is the subunit of brain glycogen synthase. Immunoreactivity of glycogen synthase was universally distributed in all regions of the brain, although hippocampus, cerebral cortex, caudatoputamen, and cerebellar cortex had relatively high immunoreactivity. Light microscopic examination revealed that the immunoreactivity was found in all cell types, such as neurons in several regions, astrocytes, ependymal cells surrounding the ventricle, oligodendrocytes, and epithelial cells of the choroid plexus in the ventricle. Immunoreactive intensity was more prominent in neurons than glial cells. Immunostaining may be a useful tool for investigation of the state of glycogen metabolism under normal and pathological conditions.     

甘油三酯介质中制备壳聚糖纳米粒工艺研究

用乳化溶剂扩散法结合离子沉淀交联法从甘油三酯介质中制备壳聚糖纳米粒,用L9(34)正交设计优选纳米粒制备的处方工艺条件,用显微镜测定纳米粒的粒径,用透射电镜观察纳米粒的形态。结果:正交设计确定纳米粒制备的最优处方工艺条件为:搅拌速度150 r.min-1,壳聚糖质量分数0.10%,壳聚糖分子量9.1万,甘油三酯与壳聚糖酸溶液体积之比200:1,制备的纳米粒平均粒径为(150±50)nm。甘油三酯介质中制备壳聚糖纳米粒工艺简便,制剂具有广泛应用前景。     

Effect of Grape Seed Proanthocyanidin Extracts on Methylmercury-Induced Neurotoxicity in Rats

As a highly toxic environmental pollutant, methylmercury (MeHg) can cause neurotoxicity in animals and humans. Considering the antioxidant property of grape seed proanthocyanidin extracts (GSPE), this study was aimed to evaluate the effect of GSPE on MeHg-induced neurotoxicity in rats. Rats were exposed to MeHg by intraperitoneal injection (4, 12 μmol/kg, respectively) and GSPE was administered by gavage (250 mg/kg) 2 h later. After a 4-week treatment, phosphate-activated glutaminase, glutamine synthetase, glutathione peroxidase and superoxide dismutase activities, glutamate, glutamine, malondialdehyde and glutathione contents in cerebral cortex were measured. Reactive oxygen species (ROS) and apoptosis were also estimated in cells. The results showed that the MeHg-induced neurotoxicity was significantly attenuated. GSPE significantly decreased the production of ROS, counteracted oxidative damage and increased the antioxidants and antioxidant enzymes activities in rats prior to MeHg exposure. Moreover, the effects on the rate of apoptotic cells and the disturbance of glutamate homeostasis were correspondingly modulated. These observations highlighted the potential of GSPE in offering protection against MeHg-induced neurotoxicity.