Microinjection manipulation decreases the expression of GABA-A receptor signaling pathway genes in mouse embryos derived using intracytoplasmic sperm injection.

BACKGROUND: The GABA-A receptor signaling pathway regulates proliferation, differentiation, apoptosis, and responses to overt DNA damage during embryonic development. METHODS: To analyze the gene expression after intracytoplasmic sperm injection (ICSI) and in in vivo mouse embryos, the global pattern of gene expression dataset, GSE23009, was obtained from the Gene Expression Omnibus database. Genes with differential expression were identified using the R software package, and RT-qPCR was performed to confirm the microarray results. RESULTS: Mouse blastocysts derived from ICSI fertilization had decreased expression of GABA-A receptor signaling pathway genes. However, the mechanisms underlying these changes were not elucidated. The gene expression of the GABA-A pathway was not significantly different between blastocysts obtained from IVF and in vivo fertilization. However, microinjection after IVF significantly reduced the expression of the GABA-A pathway gene to levels similar to those in the ICSI group. CONCLUSION: Based on our results, decreased gene expression is a result of the microinjection manipulation performed during ICSI.

A Systematic Review of the Efficacy and Safety of Microneedling in the Treatment of Melasma.

BACKGROUND: Melasma is an acquired disorder of hyperpigmentation that is often recalcitrant to current therapies. Microneedling is used to treat scars, striae, and rhytides and has a relatively low risk of post-treatment dyspigmentation. Several studies have examined its use in melasma. OBJECTIVE: To review the published evidence on the efficacy and safety of microneedling in the treatment of melasma. METHODS: A systematic review was performed. A meta-analysis could not be performed because of methodological differences across studies and data heterogeneity. RESULTS: Eight studies were included for analysis. Most studies assessed the utility of microneedling in combination with other topical therapies and detected some success. However, microneedling-mediated transdermal delivery of medications is not superior to microinjections of medications. There is less evidence supporting the use of microneedling as monotherapy. Microneedling, when used with a 1064-nm Q-switched Nd:YAG laser, may provide additional benefit, although with a risk of post-treatment dyspigmentation. CONCLUSION: Based on low-quality evidence, microneedling may play a role in the treatment of melasma, with the mechanism of action likely being the facilitation of delivery of topical therapies to the epidermis and dermis, and one ancillary benefit of this approach being the very low risk of postinflammatory hyperpigmentation.

Microinjection induces changes in the transcriptome of bovine oocytes.

Gene knockdown techniques are widely used to examine the function of specific genes or proteins. While a variety of techniques are available, a technique commonly used on mammalian oocytes is mRNA knockdown by microinjection of small interfering RNA (siRNA), with non-specific siRNA injection used as a technical control. Here, we investigate whether and how the microinjection procedure itself affects the transcriptome of bovine oocytes. Injection of non-specific siRNA resulted in differential expression of 119 transcripts, of which 76 were down-regulated. Gene ontology analysis revealed that the differentially regulated genes were enriched in the biological processes of ATP synthesis, molecular transport and regulation of protein polyubiquitination. This study establishes a background effect of the microinjection procedure that should be borne in mind by those using microinjection to manipulate gene expression in oocytes.

Physical drug delivery enhancement for aged skin, UV damaged skin and skin cancer: Translation and commercialization.

This review analyses physical drug delivery enhancement technologies with a focus on improving UV damaged skin, actinic keratoses and non-melanoma skin cancer treatment. In recent years, physical drug delivery enhancement has been shown to enhance cosmeceutical and skin cancer treatment efficacy, but there are pros and cons to each approach which we discuss in detail. Mechanisms of action, clinical efficacy, experimental design, outcomes in academic publications, clinical trial reports and patents are explored to evaluate each technology with a critical, translation focused lens. We conclude that the commercial success of cosmeceutical applications, e.g. microneedles, will drive further innovation in this arena that will impact how actinic keratoses and non-melanoma skin cancers are clinically managed.

Evaluation of the clinical impact of repeat application of hydrogel-forming microneedle array patches.

Hydrogel-forming microneedle array patches (MAPs) have been proposed as viable clinical tools for patient monitoring purposes, providing an alternative to traditional methods of sample acquisition, such as venepuncture and intradermal sampling. They are also undergoing investigation in the management of non-melanoma skin cancers. In contrast to drug or vaccine delivery, when only a small number of MAP applications would be required, hydrogel MAPs utilised for sampling purposes or for tumour eradication would necessitate regular, repeat applications. Therefore, the current study was designed to address one of the key translational aspects of MAP development, namely patient safety. We demonstrate, for the first time in human volunteers, that repeat MAP application and wear does not lead to prolonged skin reactions or prolonged disruption of skin barrier function. Importantly, concentrations of specific systemic biomarkers of inflammation (C-reactive protein (CRP); tumour necrosis factor-α (TNF-α)); infection (interleukin-1ß (IL-1ß); allergy (immunoglobulin E (IgE)) and immunity (immunoglobulin G (IgG)) were all recorded over the course of this fixed study period. No biomarker concentrations above the normal, documented adult ranges were recorded over the course of the study, indicating that no systemic reactions had been initiated in volunteers. Building upon the results of this study, which serve to highlight the safety of our hydrogel MAP, we are actively working towards CE marking of our MAP technology as a medical device.

Small damage of brain parenchyma reliably triggers spreading depolarization.

Objectives: Spreading depolarization (SD) is a well-recognized component of the stress response of the cortex to its acute injury. Cortical SD has been shown to occur in severe brain insults and standard neurosurgical procedures in patients and is supposed to promote delayed secondary brain injuries. Stereotactic surgery and site-specific intracerebral microinjections produce a small tissue injury when a thin needle is inserted directly into the brain parenchyma (via the cannula guide). The present study was designed to examine whether such a parenchymal damage can trigger SD.Methods: Experiments were performed in awake freely moving rats with simultaneous video-monitoring of behavior and recording of SD-related DC potentials in the cortex and striatum. A parenchymal damage was produced by 1-mm protruding of thin (0.3-mm diameter) cannula beyond the tip of cannula guide preliminary implanted into the amygdala or deep cortical layers.Results: We found that the micro-injury of the brain parenchyma the volume of which did not exceed 0.3 mm3 was sufficient to initiate SD in a very high proportion of rats (75-100%). The amygdala showed increased resistance against the injury-induced SD compared to the cortex. We further showed that SD triggered by the local micro-injury invaded remote intact regions of the cortico-striatal system and evoked specific changes in spontaneous animal behavior.Discussion: The findings indicate that SD may represent a previously unidentified side effect of local parenchymal injury during site-specific microinjections and stereotactic surgery.

Development of novel double-decker microneedle patches for transcutaneous vaccine delivery.

Microneedle (MN) patches have great potential as transcutaneous vaccine delivery devices because MNs can effectively deliver vaccine antigen into the skin through the micropores formed in the stratum corneum by low-invasive and painless skin puncturing. This study aims to develop novel double-decker MN patches which have not only high safety and efficacy but also broad applicability to various vaccine antigens. We developed two types of MN patches (PGA-MN and Nylon-MN) that are made from polyglycolic acid and Nylon-6. In pre-clinical studies, both MN patches could demonstrably deliver antigens into resected human dermal tissue, prolong antigen deposition and increase antigen-specific IgG levels after vaccination compared with conventional injections. We demonstrated both MN patches could be safely applied to human skin because no broken MNs or significant skin irritation were observed after applications in the clinical research. PGA-MN was suggested to be superior to Nylon-MN regarding human skin puncturability based on measurements of transepidermal water loss and needle failure force. A high content of tetravalent influenza hemagglutinin antigens loaded on PGA-MN could stably maintain HA titers at 35°C for 1year. Overall, double-decker MN patches can reliably and safely puncture human skin and are promising as effective transcutaneous vaccine delivery devices.

Repeat application of microneedles does not alter skin appearance or barrier function and causes no measurable disturbance of serum biomarkers of infection, inflammation or immunity in mice in vivo.

We address, for the first time, the impact of skin insertion on multiple occasions of polymeric microneedle arrays in an animal model in vivo. Dissolving microneedle arrays prepared from aqueous blends of 20% w/w Gantrez® S-97 BF and 40% w/w poly(vinyl pyrrolidone) 58kDa and hydrogel-forming microneedle arrays prepared from aqueous blends of and poly(ethyleneglycol) 10kDa were repeatedly applied to the skin of hairless mice in vivo. Skin appearance and skin barrier function, as illustrated by measurement of transepidermal water loss, were not measurably altered during the entire study period. Biomarkers of infection, immunity and inflammation/irritation were also statistically unchanged, regardless of the microneedle formulation, needle density or number of applications. Mice remained healthy throughout and continued to gain weight during the study. For example, transepidermal water loss values were typically in the range 10-15gm-2h-1 immediately prior to microneedle insertion and 15-25gm-2h-1 immediately following microneedle removal, regardless of when they were measured during the study periods. Serum biomarker levels, measured immediately post-mortem were always in the range 10-20µgml-1 for C-reactive protein, 0.5-1.5mgml-1 for Immunoglobulin G and 1000-2500pgml-1 for interleukin 1-ß and were never statistically different from untreated controls. No measurable levels of tumour necrosis factor-α were found in any animals. These findings are encouraging for the formulations investigated, suggesting that their repeated use by patients will not cause undesirable side-effects. By beginning to address potential regulatory questions at an early stage, the microneedles field will be ideally-placed to take advantage of the potential market. This work illustrates a potential pre-clinical strategy for development of regulatory dossiers on microneedle technologies.

Considerations in the use of microneedles: pain, convenience, anxiety and safety.

Transdermal delivery using microneedles is gaining increasing attention from pharmaceutical and cosmetic companies as one of the promising drug delivery methods. Microneedle products have recently become available on the market, and some of them are under evaluation for efficacy and safety. To be available in the market for cosmetic and therapeutic use, several factors should be considered, including pain, anxiety, convenience and safety. These factors are summarized and reviewed in this article according to type of microneedle. Various kinds of materials have been used for manufacturing microneedles and developing drug formulations for microneedles. Safety information about materials used for microneedles is summarized in terms of type of microneedles. In addition to their biocompatibility, mechanical safety is also discussed. This review can provide guidelines for designing microneedle products for proper use.

Transdermal Delivery of Iron Using Soluble Microneedles: Dermal Kinetics and Safety.

Currently, the iron compounds are administered via oral and parenteral routes in patients of all ages, to treat iron deficiency. Despite continued efforts to supplement iron via these conventional routes, iron deficiency still remains the most prevalent nutritional disorder all over the world. Transdermal replenishment of iron is a novel, potential approach of iron replenishment. Ferric pyrophosphate (FPP) was found to be a suitable source of iron for transdermal replenishment. The safety of FPP was assessed in this project by challenging the dermal fibroblast cells with high concentration of FPP. The cell viability assay and reactive oxygen species assay were performed. The soluble microneedle array was developed, incorporated with FPP and the kinetics of free iron in the skin; extracellular fluid following dermal administration of microneedle array was investigated in hairless rats. From the cell based assays, FPP was selected as one of the potential iron sources for transdermal delivery. The microneedles were found to dissolve in the skin fluid within 3 hours of administration. The FPP concentration in the dermal extracellular fluid declined after complete dissolution of the microneedle array. Overall, the studies demonstrated the safety of FPP for dermal delivery and the feasibility of soluble microneedle approach for transdermal iron replenishment therapy.

Development of a drug-coated microneedle array and its application for transdermal delivery of interferon alpha.

Interferon alpha (IFNα) is one of the most famous drugs for the treatment of chronic hepatitis C and various types of human malignancy. Protein drugs, including IFNα, are generally administered by subcutaneous or intramuscular injection due to their poor permeability and low stability in the bloodstream or gastrointestinal tract. Therefore, in the present study, novel IFNα-coated polyvinyl alcohol-based microneedle arrays (IFNα-MNs) were fabricated for the transdermal delivery of IFNα without the painful injection. IFNα was rapidly released from MNs in phosphate buffered solution and these MNs presented piercing ability in the rat skin. Slight erythema and irritation were observed when MNs were applied to the rat skin, but these skin damages completely disappeared within 24 h after removing the IFNα-MNs. Furthermore, the pharmacokinetic parameters of IFNα-MNs were similar to those of IFNα subcutaneous administration. Finally, IFNα-MNs showed a significant antitumor effect in tumor bearing mice similar to that of IFNα subcutaneous administration. These results indicate that IFNα-MNs are a useful biomaterial tool for protein drug therapy and can improve the quality of life in patients by avoidance of painful injections.

The ICSI procedure from past to future: a systematic review of the more controversial aspects.

BACKGROUND: ICSI is currently the most commonly used assisted reproductive technology, accounting for 70-80% of the cycles performed. This extensive use, even excessive, is partly due to the high level of standardization reached by the procedure. There are, however, some aspects that deserve attention and can still be ameliorated. The aim of this systematic review was to evaluate the results of available publications dealing with the management of specific situations during ICSI in order to support embryologists in trying to offer the best laboratory individualized treatment. METHODS: This systematic review is based on material obtained by searching PUBMED between January 1996 and March 2015. We included peer-reviewed, English-language journal articles that have evaluated ICSI outcomes in the case of (i) immature oocytes, (ii) oocyte degeneration, (iii) timing of the various phases, (iv) polar body position during injection, (v) zona-free oocytes, (vi) fertilization deficiency, (vii) round-headed sperm, (viii) immotile sperm and (ix) semen samples with high DNA fragmentation. RESULTS: More than 1770 articles were obtained, from which only 90 were specifically related to the issues developed for female gametes and 55 for the issues developed for male gametes. The studies selected for this review were organized in order to provide a guide to overcome roadblocks. According to these studies, the injection of rescue metaphase I oocytes should be discouraged due to poor clinical outcomes and a high aneuploidy rates; laser-assisted ICSI represents an efficient method to solve the high oocyte degeneration rate; the optimal ICSI timing and the best polar body position during the injection have not been clarified; injected zona-free oocytes, if handled carefully, can develop up to blastocyst stage and implant; efficient options can be offered to patients who suffered fertilization failure in previous conventional ICSI cycles. Most controversial and inconclusive are data on the best method to select a viable spermatozoa when only immotile spermatozoa are available for ICSI and, to date, there is no reliable approach to completely filter out spermatozoa with fragmented DNA from an ejaculate. However, most of the studies do not report essential clinical outcomes, such as live birth, miscarriage and fetal abnormality rate, which are essential to establish the safety of a procedure. CONCLUSIONS: This review provides the current knowledge on some controversial technical aspects of the ICSI procedures in order to improve its efficacy in specific contexts. Notwithstanding that embryologists might benefit from the approaches presented herein in order to improve ICSI outcomes, this area of expertise still demands a greater number of well-designed studies, especially in order to solve open issues about the safety of these procedures.

Analyses of the cell mechanical damage during microinjection.

The microinjection is an essential technique to introduce foreign materials into biological cells. The soft cell is inevitably ruptured by the microinjector during microinjection. We discuss the way to reduce the mechanical damage by analyzing the control parameters during microinjection. The computational model is developed with the dissipative particle dynamics to simulate the soft mechanical properties of biological cells. The cell model contains the membrane networks, the internal cytoskeleton, crosslink proteins, motors and their functions. The weak power law rheology verifies our computational model. The number of ruptured bonds is used to describe the extent of the mechanical damage that the cell experiences during microinjection. Some experiments are conducted on the Zebrafish embryos. Both the simulation works and experimental results show that the size, shape of the microinjector tip, and the injection velocity have a significant influence on the cell damage. A small, sharp microinjector with a high velocity can reduce the mechanical damage.

An arched micro-injector (ARCMI) for innocuous subretinal injection.

Several critical ocular diseases that can lead to blindness are due to retinal disorders. Subretinal drug delivery has been developed recently for the treatment of retinal disorders such as hemorrhage because of the specific ocular structure, namely, the blood retinal barrier (BRB). In the present study, we developed an Arched Micro-injector (ARCMI) for subretinal drug delivery with minimal retinal tissue damage. ARCMIs were fabricated using three major techniques: reverse drawing lithography, controlled air flow, and electroplating. In order to achieve minimal retinal tissue damage, ARCMIs were fabricated with specific features such as a 0.15 mm(-1) curvature, 45° tip bevel, 5 mm length, inner diameter of 40 µm, and an outer diameter of 100 µm. These specific features were optimized via in-vitro experiments in artificial ocular hemispherical structures and subretinal injection of indocyanine green in porcine eye ex-vivo. We confirmed that the ARCMI was capable of delivering ocular drugs by subretinal injection without unusual subretinal tissue damage, including hemorrhage.

Microneedles for intradermal and transdermal drug delivery.

The formidable barrier properties of the uppermost layer of the skin, the stratum corneum, impose significant limitations for successful systemic delivery of broad range of therapeutic molecules particularly macromolecules and genetic material. Microneedle (MN) has been proposed as a strategy to breach the stratum corneum barrier function in order to facilitate effective transport of molecules across the skin. This strategy involves use of micron sized needles fabricated of different materials and geometries to create transient aqueous conduits across the skin. MN, alone or with other enhancing strategies, has been demonstrated to dramatically enhance the skin permeability of numerous therapeutic molecules including biopharmaceuticals either in vitro, ex vivo or in vivo experiments. This suggested the promising use of MN technology for various possible clinical applications such as insulin delivery, transcutaneous immunisations and cutaneous gene delivery. MN has been proved as minimally invasive and painless in human subjects. This review article focuses on recent and future developments for MN technology including the latest type of MN design, challenges and strategies in MNs development as well as potential safety aspects based on comprehensive literature review pertaining to MN studies to date.

Effect of the site of assisted hatching on vitrified-warmed blastocyst transfer cycles: a prospective randomized study.

PURPOSE: To assess the effect of assisted hatching (AH) site on the clinical outcomes in vitrified-warmed blastocyst transfer cycles. METHODS: A total of 160 women who underwent vitrified-warmed blastocyst transfer cycles were randomized to either the ICM group (AH performing at the site near the inner cell mess, ICM), or the TE group (AH performing at the site opposite to the ICM). AH with laser zona drilling was performed 20 or 30 min after thawing once the ICM can be detected. Clinical pregnancy rate, implantation rate, live birth rate and the occurrence rate of monozygotic twins (MZT) pregnancy after transfer of these two groups were compared. RESULTS: No significant difference was found in the clinical pregnancy rate (63.8% vs. 67.5%), implantation rate (51.7% vs. 53.6%) and live birth rate (57.5% vs. 62.5%) between the ICM group and the TE group. The occurrence rate of MZT was comparable between the two groups (3.9% vs. 5.6%). CONCLUSIONS: The site of assisted hatching has no influence on the implantation, pregnancy and live birth rate in human vitrified-warmed blastocyst transfer cycles.

Optimizing gene transfer to conventional outflow cells in living mouse eyes.

The mouse eye has physiological and genetic advantages to study conventional outflow function. However, its small size and shallow anterior chamber presents technical challenges to efficient intracameral delivery of genetic material to conventional outflow cells. The goal of this study was to optimize methods to overcome this technical hurdle, without damaging ocular structures or compromising outflow function. Gene targeting was monitored by immunofluorescence microscopy after transduction of adenovirus encoding green fluorescent protein driven by a CMV promoter. Guided by a micromanipulator and stereomicroscope, virus was delivered intracamerally to anesthetized mice by bolus injection using a 33 gauge needle attached to Hamilton syringe or infusion with glass micropipette connected to syringe pump. The total number of particles introduced remained constant, while volume of injected virus solution (3-10 µl) was varied for each method and time of infusion (3-40 min) tested. Outflow facility and intraocular pressure were monitored invasively using established techniques. Unlike bolus injections or slow infusions, introduction of virus intracamerally during rapid infusions (3 min) at any volume tested preferentially targeted trabecular meshwork and Schlemm's canal cells, with minimal transduction of neighboring cells. While infusions resulted in transient intraocular pressure spikes (commensurate with volume infused, Δ40-70 mmHg), eyes typically recovered within 60 min. Transduced eyes displayed normal outflow facility and tissue morphology 3-6 days after infusions. Taken together, fast infusion of virus solution in small volumes intracamerally is a novel and effective method to selectively deliver agents to conventional outflow cells in living mice.

Safety of a novel microneedle device applied to facial skin: a subject- and rater-blinded, sham-controlled, randomized trial.

OBJECTIVE: To assess the safety of a novel microneedle device on facial skin of healthy individuals of all Fitzpatrick skin types. DESIGN: Subject- and live rater­blinded, sham-controlled, randomized trial. SETTING: University-based ambulatory dermatology service providing both primary and referral care. PARTICIPANTS: Healthy adults recruited from postings. INTERVENTION: Device or sham applied with finger pressure to the right or left sides, respectively, of the participants' lateral forehead, temple, and nasolabial fold. At the 24-hour visit, a larger area (3 × 3 matrix) at the central forehead was treated with the device, and the participants applied the device to their chins. MAIN OUTCOME MEASURE: Live blinded rater determination of local skin reaction scores (SRSs). RESULTS: At the 5-minute skin assessment, the median SRS was 1 for all skin type and age groups. There was no median pain score higher than 1 for any age or skin type group. For the sham device, median SRSs were 0 at all time points for all age and skin type groups. Mean SRSs for the device and sham were significantly different only for the lateral forehead at 5 and 30 minutes (P = .04). CONCLUSIONS: The microneedle device appears to be safe and well tolerated in both sexes and various skin types and ages. Facial skin application of the device elicits mild, self-limited, and rapidly resolving erythema marginally greater than that associated with the sham control.

Repeated microinjections into the medial prefrontal cortex (mPFC) impair extinction of conditioned place preference in mice.

The medial prefrontal cortex (mPFC) is important for extinction of many behaviors including conditioned place preference (CPP). We examined the effects of intra-mPFC inactivation (with bupivacaine) on extinction of ethanol-induced CPP in mice. Injections of both bupivacaine and vehicle impaired extinction whereas no-surgery control mice extinguished normally. Consistent with recently reported effects of mPFC lesions, these data suggest that extinction was impaired by excessive mPFC damage induced by repeated intracranial infusions.