Study of the substructure of the Morgagni and Brunescens cataract with the TAO non-coating technique

Lens tissue from a Morgagni cataract was examined by SEM and TEM. For SEM, after prefixation with glutaraldehyde and postfixation with the tannic acid/arginine/OsO₄ non-coating (TAO) technique, and for TEM, after prefixation with glutaraldehyde, postfixation with OsO₄/K₄Fe(CN)₆ and poststaining with uranyl acetate/lead citrate. The TAO technique seems to be a particularly suitable postfixation method for the SEM investigation of cararact tissue because of the presence of the protein structures present. The cortical region showed areas of radially, instead of concentrically, arranged lens fibres, degenerated lens fibres with holes (vacuoles), broken ball and socket connections between the lens fibres, and oval or spherical structures varying in size from 0.5–20 m, the largest resembling a golfball, arising from the cytoplasm of degenerating lens fibres. The smallest, 0.2–0.5 m, appear to have been expelled from the furrowed lens epithelium.     

Deconvoluting the dual hypoglycemic effect of wedelolactone isolated from Wedelia calendulacea: investigation via experimental validation and molecular docking

Wedelia calendulacea has a long history of use in the Indian Ayurvedic System of Medicine for the treatment, prevention, and cure of a diverse range of human diseases such as diabetes obesity, and other metabolic diseases. A wide range of chemical constituents, such as triterpenoid saponin, kauren diterpene, and coumestans, has been isolated from the plant. Conversely, no published literature is available in relation to the isolation of wedelolactone (WEL) for its anti-diabetic effect. The aim of the present study was to isolate the bioactive phyto-constituent from Wedelia calendulacea and to scrutinize the antidiabetic effect with its possible mechanism of action. The structure of the isolated compound was elucidated by different spectroscopy techniques. Proteins, such as dipeptidyl peptidase-4 (DPPIV), glucose transporter 1 (GLUT1), and peroxisome proliferator-activated receptors-γ (PPARγ), were also subjected to in silico docking. Later, this isolated compound was scrutinized against α-glucosidase and α-amylase enzyme activity along with an oral glucose tolerance test (OGTT) for estimation of glucose utilization. Streptozotocin (STZ) was used for the induction of type II diabetes mellitus (DM) in Wistar rats. The rats were divided into different groups and received the WEL (5, 10, and 20 mg kg⁻¹, b.w.) and glibenclamide (2.5 mg kg⁻¹, b.w.) for 28 days. The blood glucose level (BGL), plasma insulin, and body weight were determined at regular time intervals. The serum lipid profile hypolipidemic effect for the different antioxidant markers and hepatic tissue markers were scrutinized along with an inflammatory mediator to deduce the possible mechanism. With the help of spectroscopy techniques, the isolated compound was identified as wedelolactone. In the docking study, WEL showed docking scores of −6.17, −9.43, and −7.66 against DPP4, GLUTI, and PRARY, respectively. WEL showed the inhibition of α-glucosidase (80.65%) and α-amylase (93.83%) and suggested an effect on postprandial hyperglycemia. In the OGTT, WEL significantly (P < 0.001) downregulated the BGL, a marker for better utilization of drugs. In the diabetes model, WEL reduced the BGL and enhanced the plasma insulin and body weight. It also significantly (P < 0.001) modulated the lipid profile; this suggested an anti-hyperlipidemia effect. WEL significantly (P < 0.001) distorted the hepatic tissue, acting as an antioxidant marker in a dose-dependent manner. WEL significantly (P < 0.001) downregulated the C-reactive protein (CRP), tumor necrosis factor alpha (TNF-α), and interleukin 6 (IL-6) level. On the basis of the available results, we can conclude that WEL can be an alternative drug for the treatment of type II DM either by inhibiting the production of inflammatory mediator or by the downregulation of oxidative stress.

Wedelia calendulacea has a long history of use in the Indian Ayurvedic System of Medicine for the treatment, prevention, and cure of a diverse range of human diseases such as diabetes obesity, and other metabolic diseases.     

Alterations in the motor neuron–renshaw cell circuit in the Sod1G93A mouse model

Motor neurons become hyperexcitable during progression of amyotrophic lateral sclerosis (ALS). This abnormal firing behavior has been explained by changes in their membrane properties, but more recently it has been suggested that changes in premotor circuits may also contribute to this abnormal activity. The specific circuits that may be altered during development of ALS have not been investigated. Here we examined the Renshaw cell recurrent circuit that exerts inhibitory feedback control on motor neuron firing. Using two markers for Renshaw cells (calbindin and cholinergic nicotinic receptor subunit alpha2 [Chrna2]), two general markers for motor neurons (NeuN and vesicular acethylcholine transporter [VAChT]), and two markers for fast motor neurons (Chondrolectin and calcitonin‐related polypeptide alpha [Calca]), we analyzed the survival and connectivity of these cells during disease progression in the Sod1^G93A mouse model. Most calbindin‐immunoreactive (IR) Renshaw cells survive to end stage but downregulate postsynaptic Chrna2 in presymptomatic animals. In motor neurons, some markers are downregulated early (NeuN, VAChT, Chondrolectin) and others at end stage (Calca). Early downregulation of presynaptic VAChT and Chrna2 was correlated with disconnection from Renshaw cells as well as major structural abnormalities of motor axon synapses inside the spinal cord. Renshaw cell synapses on motor neurons underwent more complex changes, including transitional sprouting preferentially over remaining NeuN‐IR motor neurons. We conclude that the loss of presynaptic motor axon input on Renshaw cells occurs at early stages of ALS and disconnects the recurrent inhibitory circuit, presumably resulting in diminished control of motor neuron firing. J. Comp. Neurol. 521:1449–1469, 2013. © 2012 Wiley Periodicals, Inc.     

Corticotropin-releasing factor receptor types 1 and 2 are differentially expressed in pre- and post-synaptic elements in the post-natal developing rat cerebellum

Corticotropin-releasing factor (CRF)-like proteins act via two G-protein-coupled receptors (CRF-R1 and CRF-R2) playing important neuromodulatory roles in stress responses and synaptic plasticity. The cerebellar expression of corticotropin-releasing factor-like ligands has been well documented, but their receptor localization has not. This is the first combination of a light microscopic and ultrastructural study to localize corticotropin-releasing factor receptors immunohistologically in the developing rat cerebellum. Both CRF-R1 and CRF-R2 were expressed in climbing fibres from early stages (post-natal day 3) to the adult, but CRF-R2 immunoreactivity was only prominent throughout the molecular layer in the posterior cerebellar lobules. CRF-R1 immunoreactivity was concentrated in apical regions of Purkinje cell somata and later in primary dendrites exhibiting a diffuse cytoplasmic appearance. In Purkinje cells, CRF-R1 immunoreactivity was never membrane bound post-synaptically in dendritic spines while CRF-R2 immunoreactivity was found on plasmic membranes of Purkinje cells from post-natal day 15 onwards. We conclude that the localization of these receptors in cerebellar afferents implies their pre-synaptic control of the release of corticotropin-releasing factor-like ligands, impacting on the sensory information being transmitted from afferents. Furthermore, the fact that CRF-R2 is membrane bound at synapses, while CRF-R1 is not, suggests that ligands couple to CRF-R2 via synaptic transmission and to CRF-R1 via volume transmission. Finally, the distinct expression profiles of receptors along structural domains of Purkinje cells suggest that the role for these receptors is to modulate afferent inputs.     

Adhesion receptors mediate efficient non-viral gene delivery.

For a variety of reasons, including production limitations, potential unanticipated side effects, and an immunological response upon repeated systemic administration, virus-based vectors are as yet not ideal gene delivery vehicles, justifying further research into alternatives. Unlike viral vectors, non-viral vectors pose minimal health risks, but to meet therapeutic requirements their efficacy needs major improvement. This goal may be accomplished by better defining the mechanism of non-viral gene delivery and exploiting specific cellular properties. Here we demonstrate that transfection of epithelial cells with lipoplexes is almost exclusively mediated by the beta1 integrin cell surface receptor. More important, we show that in general, adhesion receptors can be exploited by lipoplexes to gain access to cells, including difficult-to-transfect primary neural stem cells and suspension cells, thereby leading to productive transfection. We propose that adhesion receptors serve as "natural" receptors for lipoplexes. As no natural cellular receptors for lipoplexes have previously been identified, our results are an important step forward in understanding the mechanisms of non-viral gene delivery. Moreover, the finding that adhesion receptors mediate efficient non-viral gene delivery paves the way for the optimization of (standard) transfection procedures as well as ex vivo gene therapy protocols using non-viral vectors.     

Surface Characteristics of Nanoparticles Determine Their Intracellular Fate in and Processing by Human Blood–Brain Barrier Endothelial Cells In Vitro

A polarized layer of endothelial cells that comprises the blood–brain barrier (BBB) precludes access of systemically administered medicines to brain tissue. Consequently, there is a need for drug delivery vehicles that mediate transendothelial transport of such medicines. Endothelial cells use a variety of endocytotic pathways for the internalization of exogenous materials, including clathrin-mediated endocytosis, caveolar endocytosis, and macropinocytosis. The different modes of endocytosis result in the delivery of endocytosed material to distinctive intracellular compartments and therewith correlated differential processing. To obtain insight into the properties of drug delivery vehicles that direct their intracellular processing in brain endothelial cells, we investigated the intracellular processing of fixed-size nanoparticles in an in vitro BBB model as a function of distinct nanoparticle surface modifications. Caveolar endocytosis, adsorptive-mediated endocytosis, and receptor-mediated endocytosis were promoted by the use of uncoated 500-nm particles, attachment of the cationic polymer polyethyleneimine (PEI), and attachment of prion proteins, respectively. We demonstrate that surface modifications of nanoparticles, including charge and protein ligands, affect their mode of internalization by brain endothelial cells and thereby their subcellular fate and transcytotic potential.     

The dynamic developmental localization of the full-length corticotropin-releasing factor receptor type 2 in rat cerebellum

Corticotropin releasing factor receptor 2 (CRF-R2) is strongly expressed in the cerebellum and plays an important role in the development of the cerebellar circuitry, particularly in the development of the dendritic trees and afferent input to Purkinje cells. However, the mechanisms responsible for the distribution and stabilization of CRF-R2 in the cerebellum are not well understood. Here, we provide the first detailed analysis of the cellular localization of the full-length form of CRF-R2 in rat cerebellum during early postnatal development. We document unique and developmentally regulated subcellular distributions of CRF-R2 in cerebellar cell types, e.g. granule cells after postnatal day 15. The presence of one or both receptor isoforms in the same cell may provide a molecular basis for distinct developmental processes. The full-length form of CRF-R2 may be involved in the regulation of the first stage of dendritic growth and at later stages in the controlling of the structural arrangement of immature cerebellar circuits and in the autoregulatory pathway of the cerebellum.     

Acute irradiation effects on morphology and function of rat submandibular glands

In this study the morphologic and functional changes were compared after irradiation (single dose, 15 Gy) of rat submandibular salivary glands. Before and 1-10 days after local irradiation of the gland region, samples of submandibular saliva were collected after stimulation by pilocarpine. At the same time-points and also 3 h postirradiation submandibular glands were carefully extirpated and prepared for histocytologic examination (LM, TEM). Maximal increase of the lag phase and decrease of the flow rate were observed 3 days after irradiation, while [K+] and [Na+] increased and decreased, respectively, from days 1 and 3 after irradiation. Morphologic changes were observed from the third hour after irradiation, were maximal 3 days after irradiation and had partially recovered by day 10. Three hours and 1 day after irradiation degranulation of convoluted granulated tubes (CGT) was observed. Three days after irradiation the most striking morphologic changes in serous and mucous cells were distension of the cisternae of the RER, degeneration of mitochondria and vacuolization of the cytoplasm. Fibril-like condensations of electron dense material in the mucous granules were observed 3 h, 1 and 6 days after irradiation. Regranulation of CGT cells was observed from day 6. From this study it is concluded that changes in salivary gland function can be observed before major morphologic changes occur. Functional changes persist after the morphologic changes seem to have virtually returned to normal.     

Low-voltage field-emission scanning electron microscopy of non-coated guinea-pig hair cell stereocilia

The stereociliar structures of the guinea-pig cochlear organ of Corti were studied at low-voltage (1–5 kV) with field-emission scanning electron microscope (SEM) using various pre- and post-fixation methods, such as OTOTO (OsO4/thiocarbohydrazide/OsO4/thiocarbohydrazide/OsO4) and TAO (tannic acid/arginine/OsO4), and different dissection procedures of the cochlea. A perfusion and immersion pre-fixation with glutaraldehyde, in combination with removal of the bony wall and stria vascularis from the cochlea, followed by the TAO non-coating treatment, gave the best result at 2 kV acceleration voltage.

Due to these new techniques, several interesting delicate structures of the stereocilia, in particular fine surface structures, were detected for the first time using SEM. These findings include the different types of cross-links and tip links, i.e., the fine surface morphology of the stereocilia and their attachments and imprints in the tectorial membrane (TM). One of the most interesting findings in this study is a network of long filamentous structures, which has been identified mainly at the top of the longest stereocilia and the undersurface of the TM and which may represent the glycocalyx. These findings and their possible implications in the process of mechanoelectrical transduction will be discussed.