Keratin films for ocular surface reconstruction: Wound healing in an in-vivo model.

The most commonly used tissue substitute for ocular surface reconstruction is human amniotic membrane (AM). Because of its low biomechanical strength and intransparency there is a need to search for alternatives of consistent quality. This study, further explored the biocompatibility of Keratin Film (KF) and its ability to sustain corneal epithelial wound healing. In three equal groups of 5 New Zeeland white rabbits a 4 mm superficial keratectomy was created in the right eye. Five eyes received a KF, five a human AM graft and the remaining five no implant. All eyes were treated with ofloxacin and dexamethasone eye drops and followed up for 10 days. Corneal fluorescein staining, vascularization, and transparency were assessed using slit lamp biomicroscopy according to a standardized grading score during and at the end of follow-up. The corneal-scleral-button was excised and processed for histology. After 10 days all eyes which had received a KF showed complete epithelial healing and no signs of neovascularization. In the AM group 1 eye showed a persistent epithelial defect at day 10 and 2 eyes showed neovascularization at day 7 resolving at day 10. Transparency improved progressively both in the KF group as well as in the AM group towards the end of the follow. Histology showed a multilayer epithelium firmly adherent to the KF with no evidence of keratocyte migration or inflammatory reaction in the corneal stroma. In this study on rabbit eyes KF better supported corneal epithelial wound healing than amniotic membrane.

Cellular uptake of coumarin-6 under microfluidic conditions into HCE-T cells from nanoscale formulations.

UNLABELLED: In vitro studies of ocular bioavailability of active pharmaceutical ingredients (API) from colloidal drug delivery systems do not consider physiological shear stress generated by eyelid wiping and tear flow. The present study introduces a live cell imaging approach which enables the investigation of model drug uptake from various formulations under shear stress by using custom-made microchannels for the cultivation of human corneal epithelial cells (HCE-T). Coumarin-6 (C-6) was used as a model API incorporated into solid lipid nanoparticles and liposomes, and as an aqueous crystalline suspension. Confocal laser scanning microscopy visualized C-6 uptake into HCE-T cells in a time-resolved manner with an applied shear stress of 0.1 Pa. Static conditions were also studied for comparative purposes. Additionally, solid lipid nanoparticles (SLN) were labeled with a fluorescent phospholipid to check whether C-6 uptake was associated with SLN incorporation into the cells. RESULTS: Intact SLN were not incorporated into the cells, i.e., C-6 was passively redistributed from SLN to lipophilic cellular compartments. C-6 was enriched up to a given limit in HCE-T cells within 5 min of contact with the dispersions both under static and under flow conditions. The C-6 delivery rate from liposomes was superior to that from SLN whereby the suspension exhibited the lowest rate. C-6 release rates were comparable for static and flow conditions. Alternate flushing with formulations and buffer revealed that cells accumulated C-6. The results suggest that combining microfluidics with live cell imaging provides a valuable option for in vitro studies of ocular drug delivery.

Does lactobionic acid affect the colloidal structure and skin moisturizing potential of the alkyl polyglucoside-based emulsion systems?

Moisturizing creams are the most prescribed products in dermatology, essential in maintaining healthy skin as well as in the topical treatment of some diseases. The irritation potential of commonly used emulsifiers and moisturizing ingredients, but also their mutual interactions, could affect the functionality and safety of those dermopharmaceutics. The aim of this study was to promote moisturizing alkyl polyglucoside (APG)-based emulsion as vehicle for lactobionic acid (LA), advantageous representative of the alphahydroxyacids (AHAs)-multifunctional moisturizers, assessing the safety for use (in vitro acute skin irritation test using cytotoxicity assay compared with in vivo data obtained using skin bioengineering methods) and in vivo moisturizing capacity (bioengineering of the skin). In order to investigate possible interactions between APG mild natural emulsifier-based emulsion and LA, a deeper insight into the colloidal structure of the placebo and the emulsion with LA was given using polarization and transmission electron microscopy, rheology, thermal and texture analysis. This study showed that APG-based emulsions could be promoted as safe cosmetic/dermopharmaceutical vehicles and carriers for extremely acidic and hygroscopic AHA class of actives (specifically LA); prospective safety for human use of both APG and LA with the correlation between in vivo and in vitro findings was shown. However, it was revealed that LA strongly influenced the colloidal structure of the emulsion based on APGs and promoted the formation of lamellar structures which reflects onto the mode of water distribution within the cream. The advantageous skin hydrating potential of LA-containing emulsion vs. placebo was unlikely to be achieved, pointing that emulsions stabilized by lamellar liquid crystalline structures probably are not satisfying carriers for highly hygroscopic actives in order to reach the full moisturizing potential. Safe and effective use on dry skin is presumed.

Efficacy and safety of paravertebral blocks in breast surgery: a meta-analysis of randomized controlled trials.

BACKGROUND: Thoracic paravertebral blocks (PVBs) are successfully performed for pain management after breast surgery. The aim of the present quantitative systematic review was to assess the efficacy and adverse events of PVB in women undergoing breast surgery. METHODS: The systematic search, data extraction, critical appraisal, and pooled analysis were performed according to the PRISMA statement. The relative risk (RR), mean difference (MD), and their corresponding 95% confidence intervals (CIs) were calculated using the RevMan statistical software for dichotomous and continuous outcomes, respectively. Pain scores were converted to a scale ranging from 0 (no pain) to 10 (worst pain). RESULTS: Fifteen randomized controlled trials (published between 1999 and 2009) including 877 patients met the inclusion criteria. There was a significant difference in worst postoperative pain scores between PVB and general anaesthesia (GA) at <2 h (MD: -2.68; 95% CI: -3.33 to -2.02; P<0.00001), 2-24 h (MD: -2.34; 95% CI: -2.42 to -1.12; P<0.00001), and 24-48 h (MD: -1.75; 95% CI: -3.19 to 0.31; P=0.02). Accordingly, lower pain scores were observed for combined PVB with GA compared with GA alone for <2 h (MD: -1.87; 95% CI: -2.53 to -1.21; P<0.00001), 2-24 h (MD: -2.21; 95% CI: -3.07 to -1.35; P<0.00001), and 24-48 h (MD: -1.80; 95% CI: -2.92 to 0.68; P=0.002). The RR for the reported adverse events (e.g. pneumothorax) was low. CONCLUSIONS: There is considerable evidence that PVB in addition to GA or alone provide a better postoperative pain control with little adverse effects compared with other analgesic treatment strategies.

[Concepts for perioperative pain therapy. A critical stocktaking]. / Konzepte zur perioperativen Schmerztherapie. Eine kritische Bestandsaufnahme.

Despite numerous publications, new guidelines for the treatment of acute pain and efforts from a number of initiatives, there is still a tremendous need for improvement in postoperative pain therapy. One of the reasons for the shortcomings in the care of patients with postoperative pain is the lack of applicability of guidelines in daily clinical practice. Therefore, simple but effective and easy to implement concepts need to be developed. In the following review, different concepts that have been developed over recent years are presented and evaluated for their effectiveness. One of these is the notion of balanced analgesia, currently probably one of the most widely used perioperative therapy concepts. The idea of this concept is to reduce the doses of analgesics, e.g. opioids, through combinations of different classes of analgesics, thereby reducing their side effects. However, recent studies and essential meta-analyses indicate pitfalls using this concept. The pros and cons will be discussed and ideas on how to deal with balanced analgesia in daily practice will be given. Another pain concept of "procedure-specific postoperative pain therapy", is an appealing idea of an international initiative from surgeons and anaesthesiologists and an essential part of the German S3 guidelines for acute pain released last year. Critical evaluation of the available recommendations for procedure-specific analgesia together with the presentation of relatively simple but evidence-based algorithms for specific procedures may help to implement this concept in clinical routine.

Parametric investigation of static and dynamic cell culture conditions and their impact on hCMEC/D3 barrier properties.

In brain research, the hCMEC/D3 cell line is widely used for the establishment of a human in vitro blood-brain barrier (BBB) model. However, its barrier integrity seems to be insufficient for drug permeability studies, represented by rather low transendothelial electrical resistance (TEER) and high permeability of small molecules. Therefore, this study covers a parametric investigation of static and dynamic cell culture conditions to improve barrier functionality of hCMEC/D3. The effect of basal media was investigated by analyzing changes in proliferation rate, barrier integrity and gene expression of cellular junction proteins. The cells were able to grow in different cell culture media, including serum-free media. However, none of these media enhanced strongly the growth rate or barrier integrity compared to the microvascular endothelial cell growth medium-2 (EGM™-2 MV). Furthermore, hCMEC/D3 cells did not respond positively regarding TEER to any tested parameter neither supplements, coating materials nor co-cultures with the human immortalized astrocyte cell line SVGmm. Furthermore, the impact of dynamic conditions was examined by using the Dynamic Micro Tissue Engineering System (DynaMiTES). Cultivation conditions were successfully adapted to the DynaMiTES design and no negative effect was detected by analyzing cell viability and cell count, albeit TEER remained also unchanged. Consequently, the hCMEC/D3 model has considerable limitations and further improvements or alternative cell lines are required.

[Long-term prognosis of Peters anomaly]. / Langzeitprognose der Peters-Anomalie.

BACKGROUND: Peters anomaly normally presents in early childhood. Common features are central corneal opacities and dysgeneses of the anterior eye segment. Early surgery is commonly warranted to prevent deep amblyopia or because of severe glaucoma. We herein present the clinical outcomes of all patients treated in the Eye Center of the Albert Ludwigs University of Freiburg since 2005. Emphasis is placed on the Peters subtype. METHODS: Data were collected retrospectively by means of chart review. Kaplan-Meier analyses were used to estimate visual prognosis, the indication for keratoplasty, and the incidence of retinal detachment. RESULTS: A total of 23 patients were identified. Subtype distribution was 40% type 1, 50% type 2, and 10% Peters plus syndrome. Ten patients were female (45%). Mean age at first presentation was 5 years; mean follow-up period censored in terms of eyeball preservation was 2 years (0 months-8 years). At mean follow-up, 40.5% of all patients had undergone at least one keratoplasty (up to six per eye); 43% had undergone glaucoma surgery (cylophotocoagulation, trabeculectomy, implants) at this time. Important complications were retinal detachment (31%) and phthisis bulbi (15%). After 4 years, visual acuity in the better eye was at most 0.05 in every second patient. CONCLUSION: Prognosis of visual acuity in Peters anomaly is poor. It is generally not possible to restore visual function in the long run, i. e., reading-grade visual acuity is rarely achieved. Surgical interventions are associated with a high risk of severe complications. Therefore, the young patients should be connected to institutions for visually impaired persons at an early stage.

[Treated cases of retinopathy of prematurity in Germany : 5-year data from the Retina.net ROP registry]. / Behandelte Frühgeborenenretinopathie in Deutschland : 5-Jahres-Daten des Retina.net ROP-Registers.

BACKGROUND: Retinopathy of prematurity (ROP) is one of the main reasons for childhood blindness. The number of infants requiring treatment, however, is low for individual centers. The Retina.net ROP registry has been founded to allow a joint analysis of treatment patterns and courses post treatment. OBJECTIVE: This paper reports treatment patterns over 5 years. MATERIAL AND METHODS: All infants born between January 2011 and December 2015 who were entered into the treatment registry by one of the 12 participating centers were analyzed. RESULTS: The data of 150 infants (292 eyes) were analyzed and ROP 3+ in zone II was the most prevalent treatment indication. Gestational age and birth weight remained stable over the years. The treatment patterns, however, changed with anti-VEGF treatment (bevacizumab or ranibizumab) accounting for only 10% of treated eyes in 2011 but for 56% and 30% in 2014 and 2015, respectively. Almost all eyes with AP-ROP or zone I disease received anti-VEGF treatment. Zone II disease was predominantly treated with laser photocoagulation. Recurrences were more common and appeared later in the anti-VEGF group compared to the laser group (23%/interval 60 days vs. 17%/interval 23 days). Perioperative complications were evenly distributed across treatment groups. CONCLUSION: The data in this analysis represent about 10-15% of treated infants in Germany. The results provide evidence for an increasing use of anti-VEGF agents for ROP. The data reflect a selection bias for anti-VEGF treatment in eyes with a more aggressive disease. This needs to be considered when interpreting data such as disease recurrence rates. The risk for late recurrences after anti-VEGF treatment is of particular clinical significance.

Development of a conjunctival tissue substitute on the basis of plastic compressed collagen.

Ocular surface disorders, such as pterygium, cicatricial pemphigoid and external disruptions, can cause severe inflammation, scarring, fornix shortening as well as ankyloblepharon. Current treatments do not resolve these conditions sufficiently. The aim of this study was to evaluate clinical applicability and suitability of plastic compressed collagen to serve as a substrate for the expansion of human conjunctival epithelial cells in order to develop an epithelialized conjunctival substitute for fornix reconstruction. Human conjunctival epithelial cells were expanded on plastic compressed collagen gels. Epithelial cell characteristics were evaluated by haematoxylin and eosin staining, electron microscopy and cytokeratin expression. The expression of putative epithelial progenitor cell markers p63α, ABCG2 and CK15 was assessed by immunostaining. The proliferative capacity and clonal growth of the cells was evaluated before (P0) and after expansion (P1) on the plastic compressed collagen gels by colony forming efficiency assay. The potential clinical applicability of this gel substitutes was evaluated by assessment of their biomechanical properties as well as their surgical handling. Human conjunctival epithelial cells cultured on plastic and plastic compressed collagen gels formed a confluent cell layer and expressed CK19. The cells showed expression of the putative epithelial progenitor cell markers p63α, ABCG2 and CK15 and sustained colony forming ability. The compressed collagen gels showed a high ultimate tensile strength and elasticity and the surgical handling of gels was comparable to amniotic membrane. An epithelialized conjunctival tissue construct on the basis of compressed collagen might therefore be a promising alternative bioartificial tissue substitute for conjunctival reconstruction. Copyright © 2015 John Wiley & Sons, Ltd.

Cartilage degradation by stimulated human neutrophils: reactive oxygen species decrease markedly the activity of proteolytic enzymes.

BACKGROUND: Although neutrophilic granulocytes clearly contribute to cartilage degradation in rheumatic diseases, it is unclear if reactive oxygen species (ROS) or proteolytic enzymes are the most important components in cartilage degradation and how they interact. RESULTS: Neutrophils were stimulated by chemicals conferring a different degree of ROS formation and enzyme release. Supernatants of neutrophils were incubated with thin slices of pig articular cartilage. Supernatants of cartilage were assayed by NMR spectroscopy, MALDI-TOF mass spectrometry and relevant biochemical methods. Stimulation conditions of neutrophils correlated well with the extent of cartilage degradation. Due to the release of different enzymes, cartilage degradation could be best monitored by NMR since mainly low-mass degradation products were formed. Astonishingly, the suppression of the formation of ROS resulted in decreased cartilage degradation. CONCLUSION: ROS formed by neutrophils are not directly involved in cartilage degradation but influence the activity of proteolytic enzymes, which are the main effectors of cartilage degradation.

Drug release and permeation studies of nanosuspensions based on solidified reverse micellar solutions (SRMS).

Solidified reverse micellar solutions (SRMS), i.e. mixtures of lecithin and triglycerides, offer high solubilisation capacities for different types of drugs in contrast to simple triglyceride systems [Friedrich, I., Müller-Goymann, C.C., 2003. Characterisation of SRMS and production development of SRMS-based nanosuspensions. Eur. J. Pharm. Biopharm. 56, 111-119]. Nanosuspensions based on SRMS were prepared by homogenisation close to the melting point of the SRMS matrix. In a first step the SRMS matrices of 1:1 (w/w) ratios of lecithin and triglycerides were loaded with 17beta-estradiol-hemihydrate (EST), hydrocortisone (HC) or pilocarpine base (PB), respectively, and subsequently ground in liquid nitrogen to minimise drug diffusion later on. The powder was then dispersed in a polysorbate 80 solution using high pressure homogenisation. The drug loading capacities of the nanosuspensions were very high in the case of poorly water-soluble EST (99% of total 0.1%, w/w, EST) and HC (97% of total 0.5%, w/w, HC) but not sufficient with the more hydrophilic PB (37-40% of total 1.0%, w/w, PB). These findings suggest SRMS-based nanosuspensions to be promising aqueous drug carrier systems for poorly soluble drugs like EST and HC. Furthermore, in vitro drug permeation from the different drug-loaded nanosuspensions was performed across human cornea construct (HCC) as an organotypical cell culture model. PB permeation did not differ from the nanosuspension and an aqueous solution whereas the permeation coefficients of HC-loaded nanosuspensions were reduced in comparison to aqueous and oily solutions of HC. However, the permeated amount was higher from the nanosuspensions due to a much lower HC concentration in the solution than that in the nanosuspension (solution 0.02%, w/w, versus nanosuspension 0.5%, w/w). The high drug load of the nanoparticles provides prolonged HC release. Permeated amounts of EST were reduced in comparison to HC and only detectable with an ELISA technique. The EST release from nanosuspensions and different EST-loaded systems revealed a prolonged EST release from the nanoparticulate systems in contrast to a faster release of an oily solution of an equal EST concentration. With regard to an aqueous EST suspension of similar concentration which represents a depot system the release rate from the nanosuspensions revealed the same order of magnitude which points again to a prolonged release potential of the nanosuspensions.

[Esterase activity of human organotypic cornea construct (HCC) as in vitro model for permeation studies]. / Esteraseaktivität eines organotypischen humanen Kornea-Konstrukts (HCC) als In-vitro-Modell für Permeationsuntersuchungen Originalien.

Organotypic cornea equivalents are used as in vitro models for permeation studies. Many ophthalmic drugs are applied as ester prodrugs to achieve a higher bioavailability. The esterase activity of three corneal human cell lines (epithelial, stromal, endothelial cells) as well as of excised porcine cornea, human donor cornea and human cornea construct (HCC) was investigated and compared. Esterase activity was determined using p-nitrophenyl acetate and hydrocortisone acetate (HCA) as esterase substrates. Hydrocortisone acetate permeation across porcine cornea, human donor cornea and HCC was studied in vitro using Franz-diffusion cells. Corneal epithelial cells showed the highest esterase activity and only small differences to keratocytes and endothelial cells were detectable. The permeation barrier properties of the different corneal tissues were very similar in the case of HCA permeation whereas HCA metabolism rates were in the ranking order of porcine cornea > HCC > human donor cornea. Permeation and metabolism studies indicate that the in vitro permeation model HCC is able to adequately convert hydrocortisone acetate to hydrocortisone.

Selective prevention of mechanical hyperalgesia after incision by spinal ERK1/2 inhibition.

BACKGROUND: Activation of extracellular signal-regulated kinases (ERK1/2) has been shown to play an important role in several pain states. Here we investigated the ERK1/2 contribution to non-evoked and evoked pain-like behaviour in rats after surgical incision. METHODS: Spinal phosphorylation of ERK1 and ERK2 was assessed 15 min, 4 h, 24 h and 5 days after plantar incision and sham incision. The effect of PD98059, a specific inhibitor of ERK1/2 activation, administered intrathecally (IT) 1 h before or 2 h after incision on spinal ERK1 and ERK2 phosphorylation was assessed. In behavioural experiments, the effect of PD98059 administered 1 h before or after incision on non-evoked pain behaviour and mechanical and heat hyperalgesia was assessed. RESULTS: Phosphorylated ERK1 and ERK2 were rapidly increased in the ipsilateral dorsal horn from rats after incision post-operatively. This increased ERK1 and ERK2 phosphorylation were blocked by PD98059 administered before incision. In congruence, IT administration of PD98059 before incision delayed mechanical hyperalgesia after incision; however, administration after incision had only a modest effect on mechanical hyperalgesia. In addition, PD98059 did not affect non-evoked pain behaviour or heat hyperalgesia after incision. CONCLUSION: The results suggest that spinal ERK1 and ERK2 are involved in regulation of pain after incision differentially with regard to the pain modality. Furthermore, blockade of ERK1/2 activation was most effective in a preventive manner, a condition which is rare after incision. Spinal ERK1/2 inhibition could therefore be a very useful tool to manage selectively movement-evoked pain after surgery in the future.

Reactions of pholasin with peroxidases and hypochlorous acid.

The ability of myeloperoxidase (MPO) and horseradish peroxidase (HRP) to induce chemiluminescence (CL) in Pholasin (Knight Scientific, Plymouth, UK), the photoprotein of the Common Piddock Pholas dactylus, was studied. The oxidation of Pholasin by compound I or II of HRP induced an intense light emission, whereas native HRP showed only a small effect. The luminescence observed upon incubation of Pholasin with native MPO was diminished by preincubation with catalase. Considering the high instability of diluted MPO, it is concluded that traces of hydrogen peroxide in water converted MPO to its active forms, compound I and/or II, which are able to oxidize Pholasin. Indeed, the addition of hydrogen peroxide to a mixture of MPO and Pholasin induced an intense burst of light. This emission was enhanced in degree and duration in the absence of chloride. Hypochlorous acid, the reaction product of Cl(-) and compound I of MPO, was itself able to elicit a luminescent response in Pholasin and this luminescence was strongly inhibited by methionine and taurine. However, both of these HOCl scavengers only slightly reduced the light emission induced by MPO/H(2)O(2) in both the presence or absence of chloride. Thus, hypochlorous acid produced by the MPO/H(2)O(2)/Cl(-) system, under the conditions described in this study, did not contribute to Pholasin luminescence. The Pholasin luminescence elicited by formyl-leucyl-methionyl-phenylalanine (fMLP)-stimulated neutrophils depends both on superoxide anion radicals and higher oxidation states of myeloperoxidase (but not on hypochlorous acid). This is shown by the inhibition of luminescence with superoxide dismutase and potassium cyanide, together with the lack of effect of both methionine and taurine. The luminescence response is about eight times greater in cells stimulated with fMLP/cytochalasin B than with fMLP alone.

The photoprotein pholasin as a luminescence substrate for detection of superoxide anion radicals and myeloperoxidase activity in stimulated neutrophils.

Pholasin, the photoprotein of the common piddock Pholas dactylus, emits an intense luminescence upon oxidation. The contribution of superoxide anion radicals and myeloperoxidase (MPO) to Pholasin luminescence in stimulated neutrophils was investigated. Data on Pholasin luminescence were compared with results of superoxide anion radical generation detected by the cytochrome c test as well as with the release of elastase and MPO. In N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulated neutrophils, most of the luminescence is caused by superoxide anion radicals, whereas MPO shows only a small effect as shown by coincubation with superoxide dismutase (SOD) as well as potassium cyanide (KCN), an inhibitor of MPO. However, both, O2- and MPO contribute to light emission in fMLP/cytochalasin B and phorbol myristoyl acetate (PMA) stimulated cells. Thus, the kinetics of O2- generation and MPO release can be very well detected by Pholasin luminescence in stimulated neutrophils. Degranulation of azurophilic granules was assessed using an ELISA test kit for released MPO or detection of elastase activity with MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide in the supernatant of stimulated cells. Both approaches revealed concurrently similar results concerning the amount and kinetics of enzyme release with data of Pholasin luminescence. Both, cytochrome c measurements and Pholasin luminescence indicate that fMLP/cytochalasin B and PMA stimulated neutrophils produce more O2- than fMLP stimulated cells. Thus, Pholasin luminescence can be used to detect, sensitively and specifically, O2- production and MPO release from stimulated neutrophils.

Human corneal equivalent as cell culture model for in vitro drug permeation studies.

AIMS: For the study of transcorneal in vitro permeation of ophthalmic drugs, excised animal cornea or corneal epithelial cell culture are frequently used as a replacement for the human cornea. The main purposes of this study were to reconstruct a complete human organotypic cornea equivalent, consisting of all three different cell types (epithelial, stromal, and endothelial); to test the barrier function of this bio-engineered human cornea using three different model drugs (pilocarpine hydrochloride (PHCl), befunolol hydrochloride (BHCl), and hydrocortisone (HC)); and to determine its usefulness as an in vitro model for prediction of ocular drug absorption into the human eye. METHODS: A multilayer tissue construct was created step by step in Transwell cell culture insert using SV-40 immortalised human endothelial and epithelial cells and native stromal cells (fibroblasts). Morphology was characterised by light microscopy using routine H&E staining. Scanning electron microscopy was used to evaluate ultrastructural features. Ocular permeation of drugs across the human cornea construct was tested using modified Franz cells and compared with data obtained from excised porcine cornea and previously described porcine cornea constructs. RESULTS: and conclusion: The cornea construct exhibited typical corneal structures such as a monolayer of hexagonally shaped endothelial cells and a multilayered epithelium consisting of seven to nine cell layers with flat superficial cells. The formation of microplicae and microvilli was also confirmed. The human cornea construct showed similar permeation behaviour for all substances compared with excised porcine cornea. However, permeability (permeation coefficients K(p)) of the human cornea equivalent (PHCl 13.4*10(-6) (SD 3.01*10(-6)); BHCl 9.88*10(-6) (SD 1.79*10(-6)); HC 5.41*10(-6) (SD 0.40*10(-6)) cm/s) was about 1.6-1.8 fold higher than excised porcine cornea. Compared with data from the porcine cornea construct the cultivated human equivalent showed a decreased permeability. The reconstructed human cornea could be appropriate to predict drug absorption into the human eye.

Cartilage degradation by hyaluronate lyase and chondroitin ABC lyase: a MALDI-TOF mass spectrometric study.

Matrix-assisted laser desorption ionization and time-of-flight mass spectrometry (MALDI-TOF MS) has been used to investigate degradation products of two selected polysaccharides of cartilage (chondroitin sulfate and hyaluronic acid). Testicular hyaluronate lyase and chondroitin ABC lyase were used for enzymic digestion of both polysaccharides as well as of cartilage specimens. Polysaccharide solutions and cartilage supernatants were assayed by positive and negative MALDI-TOF MS. Especially chondroitin ABC lyase produced high amounts of digestion products (unsaturated di- and tetrasaccharides) from polysaccharides as well as from cartilage, clearly monitored by MALDI-TOF MS. It is concluded that MALDI-TOF MS provides a precise and fast tool for the determination of oligosaccharides since no previous derivatization is required.

[Development of an organotypic corneal construction as an in vitro model for permeability studies]. / Entwicklung eines organotypischen Korneakonstruktes als ein In-vitro-Modell für Permeationsstudien.

BACKGROUND: In vitro investigations of transcorneal permeation behaviour with new drugs in ophthalmology are mainly carried out using excised corneas taken from slaughtered or experimental animals. Analogous to previously used dermis models, an in vitro model was constructed from porcine corneal cell cultures and the permeation barrier properties were tested and compared with permeation data from excised corneas. METHODS: Epithelial, stroma and endothelial cells were successfully isolated by treatment with various enzymes and a corneal equivalent was created step-by-step which morphologically resembled the original tissue. Five different drug formulations were investigated and pilocarpine hydrochloride was chosen as the model drug. The permeation studies were made with a modified Franz cell and analysis was performed by high performance liquid chromatography. Permeation data from excised corneas and from the cornea construct were compared whereby data obtained with lipophilic preparations did not differ (factor of 1) and those obtained with aqueous formulations were relatively similar (factor of 3-4). RESULTS AND CONCLUSIONS: The results obtained show that the cornea construct can be used as an alternative to excised corneas for in vitro investigations of ophthalmic drug preparations.

Cultivation and characterization of a bovine in vitro model of the cornea.

The aim of this study was to develop an in vitro model of the cornea of bovine cells, to characterise the model by histochemical methods and to investigate permeation of ophthalmic drugs through the model. As in the in vivo situation, an in vitro model of the cornea should consist of all three different types of cells. In the current study, the construction of the in vitro cornea was performed using cells prepared from primary cultures. To investigate the state of the cells in the cultures, growth curves were established. Immunocytochemical determination of keratin and vimentin was performed for all three isolated and sub-cultivated cell types of the bovine cornea. To further simulate the in vivo conditions, corneal epithelial cells were seeded onto the collagen-gel base containing the stromal cells with an underlying sheet of endothelium. Permeation experiments were performed with pilocarpine hydrochloride and timolol hydrogen maleate as model drugs and excised bovine cornea and the in vitro cornea as permeation barriers. The immunohistochemical investigations show that excised bovine cornea and the in vitro model of the cornea are comparable with respect to the expression of keratin K3, indicating that the primarily isolated cells correspond to the different cell types of the cornea. Culturing of the epithelial cells on the complex basis has led to the formation of a corneal epithelium with several layers, closely resembling the morphology of the in vivo epithelium. Although the permeation rates of the drug through the in vitro cornea were always higher, the sequence in which the drugs permeate through the two types of barriers was the same. The drug permeation through the in vitro cornea may therefore be a useful predictive tool to estimate the permeability coefficients of drugs through excised cornea.

Is intraoperative dexmedetomidine a new option for postoperative pain treatment? A meta-analysis of randomized controlled trials.

In the present meta-analysis, we assessed the efficacy and safety of intravenous administration of dexmedetomidine (DEX) compared with placebo or opioids for acute postoperative pain treatment in adults undergoing surgery. The meta-analysis was performed according to the preferred reporting items for systematic reviews and meta-analyses (PRISMA) statement and the recommendations of the Cochrane Collaboration. Randomized controlled trials investigating perioperative administration of DEX were included. For dichotomous outcomes relative risks (RR; 95% confidence intervals [CI]) and for continuous outcomes mean differences (MD; 95% CI) were calculated. Twenty-eight randomized controlled trials including 1420 patients were finally included. Patients treated with DEX reported lower postoperative pain intensity (MD1h postoperatively: -1.59U (numeric rating scale: 0 to 10) 95% CI: -2.37 to -0.82; P=.000001) and showed a lower postoperative opioid consumption (MD24h postoperatively: -17.24mg; 95% CI: -24.38 to -10.10; P=.00001) compared with placebo. Additionally, the DEX group showed a lower RR for opioid-related adverse events (e.g. RRNausea (postanesthesia care unit): 0.66; 95% CI: 0.43 to 1.02; P=.06). The most common adverse event in patients treated with DEX was intraoperative bradycardia with a RR of 2.66 (RR: 2.66; 95% CI: 1.54 to 4.58; P=.0004) compared with placebo. There is evidence that DEX administration leads to lower postoperative pain, reduced opioid consumption, and a lower risk for opioid-related adverse events. The comparison of DEX vs opioids for postoperative pain treatment is less clear due to limited data. The most common adverse event was intraoperative bradycardia after DEX administration. Therefore cautions in patients at risk are warranted, and large trials focusing on long-term outcomes after intraoperative DEX use are needed.