Carla Kruse

Alternative Polyadenylation and miR-34 Family Members Regulate Tau Expression

Tau pathologically aggregates in Alzheimers disease, and evidence suggests that reducing tau expression may be safe and beneficial for the prevention or treatment of this disease. We sought to examine the role of the 3′‐untranslated region (3′‐UTR) of human tau mRNA in regulating tau expression. Tau expresses two 3′‐UTR isoforms, long and short, as a result of alternative polyadenylation. Using luciferase reporter constructs, we found that expression from these isoforms is differentially controlled in human neuroblastoma cell lines M17D and SH‐SY5Y. Several microRNAs were computationally identified as candidates that might bind the long, but not short, tau 3′‐UTR isoform. A hit from a screen of candidates, miR‐34a, was subsequently shown to repress the expression of endogenous tau protein in M17D cells. Conversely, inhibition of endogenously expressed miR‐34 family members leads to increased endogenous tau expression. In addition, through an unbiased screen of fragments of the human tau 3′‐UTR using a luciferase reporter assay, we identified several other regions in the long tau 3′‐UTR isoform that contain regulatory cis‐elements. Improved understanding of the regulation of tau expression by its 3′‐UTR may ultimately lead to the development of novel therapeutic strategies for the treatment of Alzheimers disease and other tauopathies.

The external microenvironment of healing skin wounds.

The skin wound microenvironment can be divided into two main components that influence healing: the external wound microenvironment, which is outside the wound surface; and the internal wound microenvironment, underneath the surface, to which the cells within the wound are exposed. Treatment methods that directly alter the features of the external wound microenvironment indirectly affect the internal wound microenvironment due to the exchange between the two compartments. In this review, we focus on the effects of temperature, pressure (positive and negative), hydration, gases (oxygen and carbon dioxide), pH, and anti‐microbial treatment on the wound. These factors are well described in the literature and can be modified with treatment methods available in the clinic. Understanding the roles of these factors in wound pathophysiology is of central importance in wound treatment.

Challenging the Conventional Therapy: Emerging Skin Graft Techniques for Wound Healing.

Background: Split-thickness skin grafting is the current gold standard for treatment of major traumatic skin loss. However, split-thickness skin grafting is limited by donor-skin availability, especially in large burns. In addition, the donor-site wound is associated with pain and scarring. Multiple techniques have been developed in the past to overcome these limitations but have been unable to achieve clinical relevance. In this study, the authors examine the novel emerging skin grafting techniques, aiming to improve the utility of split-thickness skin grafting. Methods: An extensive literature review was conducted on PubMed, MEDLINE, and Google Scholar to look for new skin grafting techniques. Special focus was given to techniques with potential for large expansion ratio and decreased donor-site pain. Results: The new modalities of modified skin grafting technique, discussed in this article, include (1) Xpansion Micrografting System, (2) fractional skin harvesting, (3) epidermal suction blister grafting, and (4) ReCell technology. These techniques are able to achieve significantly increased expansion ratios compared with conventional split-thickness skin grafting and also have decreased donor-site morbidity. Conclusions: These techniques can be used separately or in conjunction with split-thickness skin grafting to overcome the associated pitfalls. Further studies and clinical trials are needed to define the utility of these procedures and where they fit into routine clinical practice.

The effect of pH on cell viability, cell migration, cell proliferation, wound closure, and wound reepithelialization: In vitro and in vivo study

Wound microenvironment plays a major role in the process of wound healing. It contains various external and internal factors that participate in wound pathophysiology. The pH is an important factor that influences wound healing by changing throughout the healing process. Several previous studies have investigated the role of pH in relation to pathogens but studies concentrating on the effects of pH on wound healing itself are inconclusive. The purpose of this study was to comprehensively and in a controlled fashion investigate the effect of pH on wound healing by studying its effect on human primary keratinocyte and fibroblast function in vitro and on wound healing in vivo. In vitro, primary human keratinocytes and fibroblasts were cultured in different levels of pH (5.5–12.5) and the effect on cell viability, proliferation, and migration was studied. A rat full‐thickness wound model was used to investigate the effect of pH (5.5–9.5) on wound healing in vivo. The effect of pH on inflammation was monitored by measuring IL‐1 α concentrations from wounds and cell cultures exposed to different pH environments. Our results showed that both skin cell types tolerated wide range of pH very well. They further demonstrated that both acidic and alkaline environments decelerated cell migration in comparison to neutral environments and interestingly alkaline conditions significantly enhanced cell proliferation. Results from the in vivo experiments indicated that a prolonged, strongly acidic wound environment prevents both wound closure and reepithelialization while a prolonged alkaline environment did not have any negative impact on wound closure or reepithelialization. Separately, both in vitro and in vivo studies showed that prolonged acidic conditions significantly increased the expression of IL‐1 α in fibroblast cultures and in wound fluid, whereas prolonged alkaline conditions did not result in elevated amounts of IL‐1 α .

Pixel Grafting: An Evolution of Mincing for Transplantation of Full-Thickness Wounds.

Background: Split-thickness skin grafting is the gold standard for treatment of major skin loss. This technique is limited by donor-site availability in large burn injuries. With micrografting, a technique where split-thickness skin graft is minced into 0.8 × 0.8-mm pieces, the authors have demonstrated an expansion ratio of 1:100 and healing comparable to that achieved with split-thickness skin grafting. Methods: In this study, the authors explore the regenerative potential of a skin graft by cutting split-thickness skin grafts to pixel size (0.3 × 0.3 mm) grafts. Wound healing was studied in full-thickness wounds in a porcine model by creating an incubator-like microenvironment using polyurethane wound chambers. Multiple wound healing parameters were used to study the outcome of pixel grafting and compare it to micrografting and nontransplanted wounds. Results: The authors’ results show that 0.3 × 0.3-mm pixel grafts remain viable and contribute to skin regeneration. The pixel graft–transplanted wounds demonstrated a faster reepithelialization rate, decreased wound contraction, and increased mechanical stability compared with nontransplanted wounds. The reepithelialization rates of the wounds were significantly increased with pixel grafting at day 6 after wounding compared with micrografting. Among the other wound healing parameters, there were no significant differences between wounds transplanted with pixel grafts and micrografts. Conclusions: Pixel grafting technique would address the most commonly encountered limitations of the split-thickness skin graft with the possibility of an even larger expansion ratio than micrografting. This technique is simple and fast and can be conducted in the operating room or in the clinic.

Development of a precise experimental burn model

BACKGROUND Porcine wounds closely mimic human wounds and are often used experimentally in burn studies. Multiple burn devices have been reported but they rarely described precise amount of heat transfer and the burn devices generally have low and varying heat capacity resulting in significant and varying temperature drop. METHODS The authors developed a customized aluminum burn device with cork insulation and high heat capacity. A thermistor probe was embedded in the device to accurately measure the temperature of the aluminum. The burn injury was inflicted by preheating the burn device to 100°C and pressing on the dorsum of pig skin for different time points ranging from 5 to 30s using standardized force of 10N on the device. With the knowledge of the heat capacity of the aluminum block and the temperature drop, the amount of heat transferred can be calculated. RESULT The temperature drop was 0°C, 1°C, 2°C, 3°C and 5°C for a wound-device contact time of 5, 10, 15, 20 and 30s, respectively. The depths of injury at 72h after burn were 0.46mm, 0.82mm, 1.21mm, 1.61mm and 1.91mm at 5, 10, 15, 20 and 30s respectively. 3.1mm represented a full thickness burn. The depth of the burn wounds significantly correlated with the heat transferred per cm2 (correlation coefficient=0.96, p-value=0.03). CONCLUSION The authors describe a simple, standardized and reproducible animal burn model using a customized burn device. The high heat capacity ensures minimal temperature drop which minimizes the variability of heat transferred with a large temperature drop. The correlation between the heat transfer and the depth of injury can facilitate standardization of burn depths in future studies.

Titanium wound chambers for wound healing research.

Standardized and reproducible animal models are crucial in medical research. Rodents are commonly used in wound healing studies since, they are easily available, affordable and simple to handle and house. However, the most significant limitation of rodent models is that the wounds heal by contraction while in humans the primary mechanisms of healing are reepithelialization and granulation tissue formation. The robust contraction results in faster wound closure that complicates the reproducibility of rodent studies in clinical trials. We have developed a titanium wound chamber for rodent wound healing research. The chamber is engineered from two pieces of titanium and is placed transcutaneously on the dorsum of a rodent. The chamber inhibits wound contraction and provides a means for controlled monitoring and sampling of the wound environment in vivo with minimal foreign body reaction. This technical report introduces two modalities utilizing the titanium chambers in rats: (1) Wound in a skin island model and, (2) Wound without skin model. Here, we demonstrate in rats how the “wound in a skin island model” slows down wound contraction and how the “wound without skin” model completely prevents the closure. The titanium wound chamber provides a reproducible standardized models for wound healing research in rodents.

Wound healing from dermal grafts containing CD34+ cells is comparable to split-thickness skin micrografts

Background: Epidermal stem cells present in the skin appendages of the dermis might be crucial in wound healing. In this study, the authors located these cells in the dermis and evaluated their contribution to full-thickness wound healing in a porcine model. Methods: Four sequentially deeper 0.35-mm-thick skin grafts were harvested from the same donor site going down to 1.4 mm in depth (layers 1 through 4). The layers were minced to 0.8 × 0.8 × 0.35-mm micrografts and transplanted (1:2) onto full-thickness porcine wounds. Healing was monitored up to 28 days and biopsy specimens were collected on days 6 and 10. Multiple wound healing parameters were used to assess the quality of healing. Results: The authors’ results showed that wounds transplanted with layer 2 (0.35 to 0.7 mm) and layer 3 (0.7 to 1.05 mm) micrografts demonstrated reepithelialization rates comparable to that of split-thickness skin graft (layer 1, 0.00 to 0.35 mm; split-thickness skin graft) at day 10. At day 28, dermal micrografts (layers 2 and 3) showed quality of healing comparable to that of split-thickness skin grafts (layer 1) in terms of wound contraction and scar elevation index. The amounts of epidermal stem cells [cluster of differentiation (CD) 34+] and basal keratinocytes (KRT14) at each layer were quantified by immunohistochemistry. Conclusions: The analysis showed that layers 2 and 3 contained the most CD34+ cells and layer 1 was the richest in KRT14+ cells. The immunohistochemistry also indicated that, by day 6, CD34+ cells had differentiated into KRT14 cells, which migrated from the grafts and contributed to the reepithelialization of the wound.

Prolyl Hydroxylase Domain-2 Inhibition Improves Skeletal Muscle Regeneration in a Male Murine Model of Obesity

Obesity leads to a loss of muscle mass and impaired muscle regeneration. In obese individuals, pathologically elevated levels of prolyl hydroxylase domain enzyme 2 (PHD2) limit skeletal muscle hypoxia-inducible factor-1 alpha and vascular endothelial growth factor (VEGF) expression. Loss of local VEGF may further impair skeletal muscle regeneration. We hypothesized that PHD2 inhibition would restore vigorous muscle regeneration in a murine model of obesity. Adult (22-week-old) male mice were fed either a high-fat diet (HFD), with 60% of calories derived from fat, or a regular diet (RD), with 10% of calories derived from fat, for 16 weeks. On day 5 following cryoinjury to the tibialis anterior muscle, newly regenerated muscle fiber cross-sectional areas were significantly smaller in mice fed an HFD as compared to RD, indicating an impaired regenerative response. Cryoinjured gastrocnemius muscles of HFD mice also showed elevated PHD2 levels (twofold higher) and reduced VEGF levels (twofold lower) as compared to RD. Dimethyloxalylglycine, a cell permeable competitive inhibitor of PHD2, restored VEGF levels and significantly improved regenerating myofiber size in cryoinjured mice fed an HFD. We conclude that pathologically increased PHD2 in the obese state drives impairments in muscle regeneration, in part by blunting VEGF production. Inhibition of PHD2 over activity in the obese state normalizes VEGF levels and restores muscle regenerative potential.

Deep Temporal Fascia Coverage of the Loading Weight in Paralytic Lagopthalmos Patients.

Introduction:Use of a weight for lagopthalmos secondary to facial nerve paralysis is the standard technique for achieving effective eyelid closure. However, because of thin and mobile skin of the eyelid and closely opposed implant, there is increased risk of complications such as implant visibility, contour deformity, and implant extrusion. Surgical Technique:The authors describe a surgical technique involving coverage of the implanted weight with contralateral deep temporal fascia. The deep temporal fascia serves as a hammock to give an interventional barrier to prevent dehiscence of the pocket and extrusion of the ocular implant. It also provides camouflage to the irregular borders of the gold weight. Results and Conclusions:Autologous coverage of gold weight with deep temporal fascia provides an effective solution to commonly associated complications with the gold weights. The use of contralateral temporal fascia preserves the ipsilateral temporalis muscle for future facial nerve reconstruction.