Summary
Assessment of various morphological parameters of the corneal subbasal nerve plexus is a valuable method of documenting the structural and presumably functional integrity of the corneal ...innervation in health and disease. The aim of this work is to establish a rapid, reliable and reproducible method for visualization of the human corneal SBP using femtosecond laser cut corneal tissue sections. Trephined healthy corneal buttons were fixed and processed using TissueSurgeon—a femtosecond laser based microtome, to obtain thick tissue sections of the corneal epithelium and anterior stroma cut parallel to the ocular surface within approximately 15 min. A near infrared femtosecond laser was focused on to the cornea approximately 70–90 μm from the anterior surface to induce material separation using TissueSurgeon. The obtained corneal sections were stained following standard immunohistochemical procedures with anti‐neuronal β‐III tubulin antibody for visualization of the corneal nerves. Sections that contained the epithelium and approximately 20–30 μm of anterior stroma yielded excellent visualisation of the SBP with minimal optical interference from underlying stromal nerves. In conclusion, the results of this study have demonstrated that femtosecond laser cutting of the human cornea offers greater speed, ease and reliability than standard tissue preparation methods for obtaining high quality thick sections of the anterior cornea cut parallel to the ocular surface.
Lay description
Human cornea is one of the most highly innervated tissues in the body. Sectioning of the cornea for the quantitative analysis of its innervation by immunohistochemistry is necessary during health and disease. Attempts to obtain frozen or paraffin‐sections of whole corneas parallel to the plane of its innervation are technically challenging and also time consuming due to the curvature of the tissue. In an effort to overcome this difficulty, here we report a contact‐free and rapid femtosecond laser based cutting method to obtain high quality surface parallel sections of the human cornea in less than 15 min. Furthermore, femtosecond based laser microtomy can be adopted easily for sectioning of all kind of biological samples including hard tissues (teeth and bone) and offers a more standardized way of tissue sectioning by avoiding the laborious processes of sample preparation and embedding thereby preventing the loss of valuable tissue sample.
PurposeThis study was designed to compare and contrast quantitative data of the human corneal sub-basal nerve plexus (SBP) evaluated by two different methods: in vivo confocal microscopy (IVCM), and ...immunohistochemical staining of ex vivo donor corneas.MethodsSeven parameters of the SBP in large-scale IVCM mosaicking images from healthy subjects were compared with the identical parameters in ex vivo donor corneas stained by β-III-tubulin immunohistochemistry. Corneal nerve fiber length (CNFL), corneal nerve fiber density (CNFD), corneal nerve branch density (CNBD), average weighted corneal nerve fiber tortuosity (CNFTo), corneal nerve connection points (CNCP), average corneal nerve single-fiber length (CNSFL), and average weighted corneal nerve fiber thickness (CNFTh) were calculated using a dedicated, published algorithm and compared.ResultsOur experiments showed significantly higher values for CNFL (50.2 vs 21.4 mm/mm
), CNFD (1358.8 vs 277.3 nerve fibers/mm
), CNBD (847.6 vs 163.5 branches/mm
), CNFTo (0.095 vs 0.081 μm
), and CNCP (49.4 vs 21.6 connections/mm
) in histologically staining specimens compared with IVCM images. In contrast, CNSFL values were higher in IVCM images than in histological specimens (32.1 vs 74.1 μm). No significant difference was observed in CNFTh (2.22 vs 2.20 μm) between the two groups.ConclusionsThe results of this study have shown that IVCM has an inherently lower resolution compared with ex vivo immunohistochemical staining of the corneal SBP and that this limitation leads to a systematic underestimation of several SBP parameters. Despite this shortcoming, IVCM is a vital clinical tool for in vivo characterization, quantitative clinical imaging, and evaluation of the human corneal SBP.
PurposeAnalysis of microstructural alterations of corneal and limbal epithelial cells in healthy human corneas and in other ocular conditions.Patients and methodsUnilateral eyes of three groups of ...subjects include healthy volunteers (G1, n=5), contact lens wearers (G2, n=5), and patients with dry eyes (G3, n=5) were studied. Imaging of basal (BC) and intermediate (IC) epithelial cells from central cornea (CC), corneal limbus (CL) and scleral limbus (SL) was obtained by in vivo confocal microscopy (IVCM). An appropriate image analysis algorithm was used to quantify morphometric parameters including mean cell area, compactness, solidity, major and minor diameter, and maximum boundary distance.ResultsThe morphometric parameters of BC and IC demonstrated no significant differences (P>0.05) between groups. Comparison between three corneal locations (CC, CL, and SL) within the groups showed significant differences (P<0.05) with mean values of cell area, compactness, solidity, and major and minor diameter of BC that increase from CC to limbus. The BC were round and regular in the central cornea (P<0.05) compared with CL and SL.ConclusionsIVCM enables high-quality confocal images from central corneal and limbal epithelium. This quantitative study demonstrated morphological differences in the basal and intermediate epithelium between limbus and central cornea, and found no differences between contact lens wearers, dry eyes, and normal subjects.
A simple and reliable HPLC method for determining impurities in eltrombopag olamine (ELO) film-coated tablets is not available. At the same time, there is no official monograph reported. The proposed ...research is targeted at the development of a stability-indicating method for determining impurities in ELO film-coated tablets and drug substances.
To develop and validate a simple and effective HPLC method for determining impurities in ELO film-coated tablets and drug substances.
All the impurities were separated using a reverse phase (RP)-HPLC system equipped with a Zorbax SB-Phenyl 150 mm × 4.6 mm, 3.5 µm, column with UV detection at 230 nm and a flow rate of 1.2 mL/min. The column temperature was maintained at 45°C.
The proposed method was validated as per current regulatory guidelines. The coefficient of correlation was found to be >0.999 for all impurities. The LOD and LOQ for ELO and all specified impurities were determined. The precision and accuracy were obtained for ELO and its related impurities. Intra- and inter-day RSD values were between 1.22 and 2.04%, and impurity recovery varied between 93.80 and 103.69%. The stability of standard and sample solutions was established for 24 h.
As per recent guidelines, a stability-indicating method has been developed to determine the impurities in ELO film-coated tablets and drug substances. QbD-based robustness was performed and proved that the method was robust.
The proposed article is the first RP-HPLC method for determining impurities in ELO film-coated tablets and drug substances. The quality by design (QbD) concept was utilized to verify the method performance.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
*equal contribution
The activation of hepatic stellate cells (HSCs) is a central pathological process in the development of liver fibrosis. Thus, activated HSCs are attractive targets for ...antifibrotic therapies. We established an adenoviral based gene transfer system for the selective transduction of HSCs by linking a HSC-specific peptide to the virus envelope. The NGFp peptide displays a part of the nerve growth factor (NGF) binding site to p75 neurotrophin receptor (p75NTR), which is in the liver solely expressed on HSCs.
Two different methods for the construction of virus particles selectively infecting HSCs were realized. Both the covalent coupling of the peptide to the fiber by polyethylene glycol (PEG) and a non-covalent linked bispecific anti fiber single chain antibody (S11) were used to modify the natural adenovirus (Ad) tropism of the native adenoviral vector (Ad.GFP; including GFP reporter gene).
In vitro studies with primary mouse hepatocytes and in vivo infection experiments with Balb/C mice showed an equivalent inhibition of the Ad tropism to coxsackie-adenovirus receptor (CAR) by PEG- as well as S11-linkage to Ads (Ad.GFP). Additionally, NGFp-coupled Ads transduced selectively primary cultured HSCs and HSCs after intravenous infection of mice livers. The latter could be verified by visualizing the colocalization of GFP fluorescence and HSC-associated vitamin A autofluorescence using intravital fluorescence microscopy and subsequent immunohistochemical analysis of the of GFP and p75NTR
The use of p75NTR as a target for redirecting adenoviral vectors to HSCs is a new strategy to modulate these cells selectively. The shift of the natural Ad tropism is possible through covalent and non-covalent linking of the NGFp to the virus envelope. This offers new possibilities to introduce therapeutic genes into HSC without any undesirable side effects on hepatocytes or other non-parenchymal liver cells.
berit.genz@uni-rostock.de
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