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.

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.

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.

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.

Parathyroid hormone (1-34)-coated microneedle patch system: clinical pharmacokinetics and pharmacodynamics for treatment of osteoporosis.

OBJECTIVES: To evaluate the clinical PK/PD of PTH(1-34) delivered by a novel transdermal drug-coated microneedle patch system (ZP-PTH) for the treatment of osteoporosis. METHODS: Phase 1 PK studies evaluated the effect of site of administration, patch wear time and dose in normal volunteers, ages 40-85 yrs. Phase 2 was conducted in post-menopausal women with osteoporosis to determine the patch dose response compared to placebo patch and FORTEO® injection. RESULTS: Phase 1 ZP-PTH patch delivery demonstrated a rapid PTH plasma pulse profile with T(max) 3 times shorter and apparent T(1/2) 2 times shorter than FORTEO®. In Phase 2, ZP-PTH 20, 30 and 40 µg doses showed a proportional increase in plasma PTH AUC. Inter-subject and intra-subject AUC variability was similar for all patch doses and comparable to injection. All patch doses produced a significant increase in spine bone mineral density. Unexpectedly, ZP-PTH also produced an early increase in hip bone mineral density, an effect not observed with the injection. CONCLUSIONS: These studies suggest that this novel ZP-PTH patch system can deliver a consistent and therapeutically relevant PTH PK profile. Based on encouraging Phase 2 safety and efficacy data, the program is advancing into a pivotal Phase 3 clinical study.

The relationship between meiotic spindle imaging and outcome of intracytoplasmic sperm injection: a retrospective study.

Meiotic spindle analysis with a non-invasive technique, the PolScope, is used to protect the meiotic spindle from damage during microinjection. To evaluate the predictive feature of PolScope, we have designed a retrospective study to analyse the correlation between the meiotic spindle visualisation with regard to spindle location and outcomes of assisted reproductive technologies (ART), including patient age, previous cycles, the number of the collected oocytes, fertilisation rates (FR), pronuclear scoring (PNS) and embryo scoring of the days from two to five. All of the data belonging to 1496 oocytes from 190 patients were statistically analysed. We found that the oocytes having PolScope visualised spindle have higher FR, and also observed that when the spindle located at 0°-30° according to the first polar body, gave the highest FR. PNS gave higher scores in the spindle visualised group, but spindle angle did not affect PNS outcomes. Although a correlation was found between spindle visualisation and developed embryo qualities, particularly at day 2 and 3, spindle angles did not affect embryo quality. We conclude that PolScope microscopy has an efficiency to estimate FR, and cleavage stage embryo development.

Harmful microinjecting practices among a cohort of injection drug users in vancouver Canada.

OBJECTIVES: We sought to identify factors associated with harmful microinjecting practices in a longitudinal cohort of IDU. METHODS: Using data from the Vancouver Injection Drug Users Study (VIDUS) between January 2004 and December 2005, generalized estimating equations (GEE) logistic regression was performed to examine sociodemographic and behavioral factors associated with four harmful microinjecting practices (frequent rushed injecting, frequent syringe borrowing, frequently injecting with a used water capsule, frequently injecting alone). RESULTS: In total, 620 participants were included in the present analysis. Our study included 251 (40.5%) women and 203 (32.7%) self-identified Aboriginal participants. The median age was 31.9 (interquartile range: 23.4-39.3). GEE analyses found that each harmful microinjecting practice was associated with a unique profile of sociodemographic and behavioral factors. DISCUSSION: We observed high rates of harmful microinjecting practices among IDU. The present study describes the epidemiology of harmful microinjecting practices and points to the need for strategies that target higher risk individuals including the use of peer-driven programs and drug-specific approaches in an effort to promote safer injecting practices.

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.

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.

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.

Adeno-associated virus-mediated ILK gene silencing in the rat NAc core.

In this study we established conditional silencing of integrin-linked kinase (ILK) expression in Sprague-Dawley rat brain by microinjection of rAAV-2-carrying, Tet-On-regulated siRNA expression cassette into nucleus accumbens (NAc) core and induction with doxycycline. We demonstrated that inhibition of ILK expression was effectively induced by administration of doxycycline for 2 weeks while ILK expression was restored after withdrawing doxycycline for 8 days. Increases in GFAP and OX42 expression were observed 5 weeks post virus injection. Importantly, inhibition of ILK expression in the NAc core had no significant effect on cell apoptosis and animal basal locomotion and stereotypical behaviors, but decreased dendritic density of medium spiny neurons. Our studies suggest that: (1) rAAV-delivered Tet-On-regulated siRNA expression can conditionally regulate gene expression in rat brain; (2) inhibition of ILK expression has no significant effect on cell apoptosis and basal locomotor and stereotypical behaviors, but decreases dendritic density; and (3) microinjection of rAAV-2 causes inflammatory response around the injection track.

In vivo assessment of safety of microneedle arrays in human skin.

Microneedle arrays are promising devices for the delivery of drugs and vaccines into or the skin. However, little is known about the safety of the microneedles. In this study we obtained insight in the ability of microneedles to disrupt the skin barrier, which was evaluated by transepidermal water loss (TEWL). We also determined the safety in terms of skin irritation (skin redness and blood flow) and pain sensation. We applied microneedle arrays varying in length and shape on the ventral forearms of 18 human volunteers. An effect of needle length was observed, as TEWL and redness values after treatment with solid microneedle arrays of 400 microm were significantly increased compared to 200 microm. The blood flow showed a similar trend. Needle design also had an effect. Assembled microneedle arrays induced higher TEWL values than the solid microneedle arrays, while resulting in less skin irritation. However, for all microneedles the irritation was minimal and lasted less than 2h. In conclusion, the microneedle arrays used in this study are able to overcome the barrier function of the skin in human volunteers, are painless and cause only minimal irritation. This opens the opportunity for dermal and transdermal delivery of drugs and vaccines.

Targeted spinal cord therapeutics delivery: stabilized platform and microelectrode recording guidance validation.

BACKGROUND/AIMS: No validated delivery technique exists for accurate, reproducible delivery of biological therapies to discrete spinal cord targets. To address this unmet need, we have constructed a stabilized platform capable of supporting physiologic mapping, through microelectrode recording, and cellular or viral payload delivery to the ventral horn. METHODS: A porcine animal model (n = 7) has been chosen based upon the inherent morphologic similarities between the human and porcine spine. Animals underwent physiologic mapping and subsequent microinjection of a green-fluorescent-protein-labeled cell suspension. Sacrifice (t = 3 h) was performed immediately following behavioral assessment. RESULTS: Histologic analysis has supported our ability to achieve localization to the ipsilateral ventral horn in the spinal cord. Complications included death due to malignant hyperthermia (n = 1), hindlimb dysfunction attributable to epidural hematoma (n = 1), and hindlimb dysfunction attributable to cord penetration (n = 2). CONCLUSIONS: These results indicate an ability to achieve accurate targeting, but the elevated incidence of neurologic morbidity will require further studies with longer follow-ups that incorporate procedural and equipment modifications that will allow for a reduced number of cord penetrations and will account for observed cardiorespiratory-associated cord movement. These initial results reinforce the challenges of translating biological restorative therapies from small to large animal models and ultimately to humans.

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.

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.

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.

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.