Decreased cardiac glutathione peroxidase levels and enhanced mandibular apoptosis in malformed embryos of diabetic rats

OBJECTIVE— To characterize normal and malformed embryos within the same litters from control and diabetic rats for expression of genes related to metabolism of reactive oxygen species (ROS) or glucose as well as developmental genes.RESEARCH DESIGN AND METHODS— Embryos from nondiabetic and streptozotocin-induced diabetic rats were collected on gestational day 11 and evaluated for gene expression (PCR) and distribution of activated caspase-3 and glutathione peroxidase (Gpx)-1 by immunohistochemistry.RESULTS— Maternal diabetes (MD group) caused growth retardation and an increased malformation rate in the embryos of MD group rats compared with those of controls (N group). We found decreased gene expression of Gpx-1 and increased expression of vascular endothelial growth factor-A (Vegf-A) in malformed embryos of diabetic rats (MDm group) compared with nonmalformed littermates (MDn group). Alterations of messenger RNA levels of other genes were similar in MDm and MDn embryos. Thus, expression of copper zinc superoxide dismutase (CuZnSOD), manganese superoxide dismutase (MnSOD), and sonic hedgehog homolog (Shh) were decreased, and bone morphogenetic protein-4 (Bmp-4) was increased, in the MD embryos compared with the N embryos. In MDm embryos, we detected increased activated caspase-3 immunostaining in the first visceral arch and cardiac area and decreased Gpx-1 immunostaining in the cardiac tissue; both findings differed from the caspase/Gpx-1 immunostaining of the MDn and N embryos.CONCLUSIONS— Maternal diabetes causes growth retardation, congenital malformations, and decreased general antioxidative gene expression in the embryo. In particular, enhanced apoptosis of the first visceral arch and heart, together with decreased cardiac Gpx-1 levels, may compromise the mandible and heart and thus cause an increased risk of developing congenital malformation.The cellular and molecular mechanisms of diabetic embryopathy are not completely clear. Previous experimental studies have suggested that the teratological impact of a diabetic environment partly depends on excess of reactive oxygen species (ROS) in the embryo (1) as a consequence of either increased free oxygen radical formation (2–4), decreased capacity of ROS-scavenging enzymes (5–9), or both. Furthermore, previous work has demonstrated that supplementation of antioxidative agents such as copper zinc superoxide dismutase (CuZnSOD) (1,2), N-acetylcysteine (10), vitamins E and C (8), and folic acid (11) in vitro, as well as butylated hydroxytoluene (12), vitamin E (13–19), vitamin C (18,20), N-acetylcysteine (21), and folic acid (11) in vivo, attenuate malformation rate and diminish markers of oxidative stress (e.g., by normalizing tissue levels of thiobarbituric acid reactive substances [15], isoprostane 8-iso-PGF_2α [22,23], and carbonylated proteins [24]).The driving cellular force behind diabetes-induced oxidative stress is likely associated with enhanced glucose metabolism (25–27) in the embryonic/fetal cells exposed to increased ambient levels of glucose. One putative primary source of reactive radical compounds is mitochondria receiving a high influx of pyruvate and oxygen and, subsequently, producing a large amount of ROS (mainly superoxide) (3) in the oxidative processes of the electron transport chain. The ensuing leakage of superoxide into other compartments of the mitochondria and the cytosol, and the further formation of hydrogen peroxide and hydroxyl radicals, should yield mitochondrial alterations (28) as well as lipid peroxidation (22) and DNA damage (29) in the embryo. There are several observations in support of this notion. The structural alterations, mainly high-amplitude swelling of the embryonic mitochondria (28), are diminished by maternal antioxidative treatment (16), thereby supporting the notion of a ROS-related etiology of the structural changes. Enhanced lipid peroxidation, as evidenced by increased levels of the isoprostane 8-iso-PGF_2α (22,23,30), may induce several teratogenic pathways in addition to the developmental disturbance caused by peroxidation of structural lipids in mitochondrial and cellular membranes. For instance, it has been demonstrated that 8-iso-PGF_2α, which is produced nonenzymatically by ROS-mediated oxidation of arachidonic acid in the offspring (30), has its own teratogenic potency (23). In addition, an excessive peroxidation of arachidonic acid may hamper prostaglandin biosynthesis by depleting precursor pools and, in particular, yield decreased concentration of prostaglandin E2 (31), which could obstruct neural tube closure (22).ROS-induced DNA damage (29) may directly disrupt development via altered expression of key genes. In addition, cellular DNA repair processes may activate poly(ADP-ribose) polymerase (PARP), which may cause glyceraldehyde-3-phosphate dehydrogenase (GAPDH) inhibition by poly(ADP-ribosylation) (32). The net result would be diminished embryonic GAPDH activity, which has been demonstrated in rat embryos subjected to diabetes in vivo and high glucose in vitro (33). Furthermore, decreased glycolytic flux proximal to GAPDH (32) and the presence of increased ambient glucose levels will yield enhanced flux in the polyol (34,35) and hexosamine (36) pathways. An increased availability of proximal glycolytic intermediaries would increase diacylglycerol production and cause activation of several protein kinase C isoforms (37,38), as well as enhance the flux in the advanced glycosylation end product pathway (39). All of the consequences of inhibited GAPDH activity may thus contribute to the teratogenic outcome of diabetic pregnancy. Evidently, there are multiple ways for a diabetes-induced state of oxidative stress in the embryo to disturb embryonic/fetal development, several of which enjoy considerable experimental support.Another consequence of a state of oxidative stress would be enhanced apoptosis in embryonic/fetal tissue (40), which has been described (26,41–43) and has been suggested to be mediated by enhanced Jun-amino-terminal kinase-1 and -2 activity (19,44). It has also been suggested that maternal diabetes induces an inflammatory state in the embryo, where proinflammatory cytokines (i.e., tumor necrosis factor-α [TNF-α] [45,46]) act to downregulate the principal ROS-scavenging enzymes via increased activity of mitogen-activated protein kinases (47). The exact relation between enhanced apoptosis and induction of malformations is still unclear, mainly since we do not fully comprehend the specific transmission of a general increase in programmed cell death into precisely restricted developmental damage to embryonic organs or organ systems (42).Based on earlier studies (7,48,49) and on a question that has been raised several times—which genes are involved in diabetes-induced embryonic dysmorphogenesis? (5,8,26,42,50)—we wanted to add to the teratological knowledge by identifying differences in gene expression between the malformed and nonmalformed offspring within the same litters of diabetic animals.Our working hypothesis was that genes of the malformed offspring with an expression pattern different from that of genes in the nonmalformed offspring within the same litter may be associated, either directly or indirectly, with the teratogenic process. We thus decided to compare gene expression of embryos that were morphologically normal and those that were malformed in litters of normal and diabetic rats. By comparing gene expression and protein distribution in embryos of the same age and with exposure to an identical intrauterine milieu, we aimed to control for possible confounding factors of the teratogenic process. From earlier work, we knew that the two tissues of the rat fetuses that are particularly vulnerable to the diabetic state are the mandible and the heart (17) (Fig. 1). We also knew that a susceptible period in diabetic rat pregnancy occurs during gestational days 6–10 (51), which corresponds to weeks 2–4 in human pregnancy. Bearing in mind that the size and relative immaturity of day-10 embryos makes them difficult to evaluate for developmental defects, we chose to interrupt the rat pregnancies on gestational day 11 in order to be able to clearly distinguish between malformed and nonmalformed embryos. We decided to relate embryonic (mal)development to expression of the major oxidative defense genes (SODs, glutathione peroxidase [Gpx]-1, Gpx-2, and catalase), to key genes of glucose metabolism (aldose reductase [AR] and GAPDH), to developmental/teratological genes [poly(ADP-ribose) polymerase (PARP)], to tumor protein 53 (p53), to bone morphogenetic protein-4 (Bmp-4), to Ret proto-oncogene (Ret), to sonic hedgehog homolog (Shh), to vascular endothelial growth factor-A (VEGF-A), to TNF-α, to interleukin-6 (IL-6), and to the tissue distribution of activated caspase-3 (denoting apoptotic rate) and Gpx-1.Open in a separate windowFIG. 1.A: Fetuses displaying micrognathia (left fetus) and normal morphology (right fetus). B: Outcome of pregnancy in the control (N) and manifestly diabetic (MD) groups, distributed as normal (□), malformed (▪), and resorbed (▒) embryos on gestational day 11. *P < 0.05 vs. N (χ² statistics). (Please see http://dx.doi.org/10.2337/db08-0830 for a high-quality digital representation of this figure.)     

Endotoxin‐induced inflammation in a rodent model up‐regulates IL‐1a expression and CD45+ leukocyte recruitment and increases the rate of reepithelialization and wound closure

Wound healing is traditionally divided into inflammation, proliferation, and remodeling phases. Several inflammatory mediators and cells regulate the inflammation phase. The specific roles for different mediators have not been clearly defined. The effects of inflammation phase modulation on wound healing were evaluated in this study. Rat full‐thickness wounds were divided into different experimental groups: (1) sterile hyper‐inflammatory wounds/endotoxin (topical endotoxin), (2) sterile hypo‐inflammatory/inhibitor group (cocktail of topical COX‐1 plus COX‐2 plus lipoxygenase inhibitors), and (3) control groups: topical saline or DMSO. After full‐thickness wound creation, custom‐made titanium chambers enclosed the wound, creating an isolated well‐controlled environment. Wound healing was followed over time; tissue biopsies and wound fluid samples were collected on days 1, 4, and 8 postoperatively. The validity of the inflammation model was confirmed by increased IL‐1a expression, increased CD45+ leukocytes recruitment in the hyper‐inflamed group as compared to the inhibitor and control groups. The reepithelialization percentage was significantly increased in the endotoxin group as compared to the inhibitor group on day 4 (60.75 vs. 22.05, p‐value <0.05) and both the inhibitor and the control group on day 8 (control group: 63.2%, inhibitor group: 28.9%, endotoxin group: 84.2%, p‐value <0.05). Also, the macroscopic wound closure was increased in the endotoxin group as compared to the inhibitor group and control group both on day 4 (control group: 69.9%, inhibitor group: 62.9%, endotoxin group: 81.9%, p‐value <0.05) and on day 8 (control group: 68.5%, inhibitor group: 69.1%, endotoxin group: 83.7%, p‐value <0.05). Endotoxin‐induced sterile inflammation up‐regulates IL‐1a expression and CD45+ leukocyte recruitment and results in faster rate of wound reepithelialization and wound closure in full‐thickness rodent wounds. Conversely, the wound reepithelialization and wound closure can be significantly delayed on treatment with a combination of cyclooxygenase and lipoxygenase inhibitors.     

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.     

A retrospective review of the incidence of various complications in different delayed breast reconstruction methods

Background: Breast reconstruction is a common standard procedure in many centres after breast cancer surgery. The aim of the present study was to investigate and compare the incidence of various complications in different reconstruction methods. Method: Six hundred and eighty-five patients were retrospectively classified into five groups: (1) Deep inferior epigastric perforator flap (DIEP), (2) latissimus dorsi flap (LD), (3) lateral thoracodorsal flap (LTDF), (4) expander with secondary implant (EXP), and (5) direct implant (DI). Demographic and perioperative data, the incidence of complications, and follow-up data were collected. Results: There was a significant difference between groups regarding overall early complications (p?<?0.001). The DIEP group had the highest incidence of overall complications (50.0%) (p?<?0.05). DIEP also had the highest incidence of fat necrosis (18.3%), skin necrosis (22.1%), and incidence of reoperation for complications (26.9%) compared to the other reconstruction methods. In the entire group of patients, the overall incidence of late complications (occurring >30 days after surgery) that needed surgical corrections was 54.7%. The incidence of late complications was 46.2% for DIEP, 66.4% for LD, 74.8% for LTDF, 44.9% for EXP, and 62.9% for DI. The DIEP group had higher incidences of late local complications (fat necrosis, skin necrosis, haematoma, seroma, and wound rupture combined), and scars requiring treatment than the other reconstruction methods. Conclusion: Meticulous registration of incidence of different complications in five different breast reconstruction methods revealed high complication rates with all methods. The differences in incidence of complications were related to the operation method used. Based on these results, careful individual planning of a breast reconstruction is mandatory.     

Man vs machine in emergency medicine – a study on the effects of manual and automatic vital sign documentation on data quality and perceived workload,using observational paired sample data and questionnaires

Background

Emergency medicine is characterized by a high patient flow where timely decisions are essential. Clinical decision support systems have the potential to assist in such decisions but will be dependent on the data quality in electronic health records which often is inadequate. This study explores the effect of automated documentation of vital signs on data quality and workload.

Methods

An observational study of 200 vital sign measurements was performed to evaluate the effects of manual vs automatic documentation on data quality. Data collection using questionnaires was performed to compare the workload on wards using manual or automatic documentation.

Results

In the automated documentation time to documentation was reduced by 6.1?min (0.6?min vs 7.7?min, p?<? 0.05) and completeness increased (98% vs 95%, p?<? 0.05). Regarding workflow temporal demands were lower in the automatic documentation workflow compared to the manual group (50 vs 23, p?<? 0.05). The same was true for frustration level (64 vs 33, p?<? 0.05). The experienced reduction in temporal demands was in line with the anticipated, whereas the experienced reduction in frustration was lower than the anticipated (27 vs 54, p?<?0.05).

Discussion

The study shows that automatic documentation will improve the currency and the completeness of vital sign data in the Electronic Health Record while reducing workload regarding temporal demands and experienced frustration. The study also shows that these findings are in line with staff anticipations but indicates that the anticipations on the reduction of frustration may be exaggerated among the staff. The open-ended answers indicate that frustration focus will change from double documentation of vital signs to technical aspects of the automatic documentation system.

     

Continuous inhibition of 11β‐hydroxysteroid dehydrogenase type I in adipose tissue leads to tachyphylaxis in humans and rats but not in mice

Background and Purpose

11β‐hydroxysteroid dehydrogenase type I (11β‐HSD1), a target for Type 2 diabetes mellitus, converts inactive glucocorticoids into bioactive forms, increasing tissue concentrations. We have compared the pharmacokinetic‐pharmacodynamic (PK/PD) relationship of target inhibition after acute and repeat administration of inhibitors of 11β‐HSD1 activity in human, rat and mouse adipose tissue (AT).

Experimental Approach

Studies included abdominally obese human volunteers, rats and mice. Two specific 11β‐HSD1 inhibitors (AZD8329 and COMPOUND‐20) were administered as single oral doses or repeat daily doses for 7–9 days. 11β‐HSD1 activity in AT was measured ex vivo by conversion of ³H‐cortisone to ³H‐cortisol.

Key Results

In human and rat AT, inhibition of 11β‐HSD1 activity was lost after repeat dosing of AZD8329, compared with acute administration. Similarly, in rat AT, there was loss of inhibition of 11β‐HSD1 activity after repeat dosing with COMPOUND‐20 with continuous drug cover, but effects were substantially reduced if a ‘drug holiday’ period was maintained daily. Inhibition of 11β‐HSD1 activity was not lost in mouse AT after continuous cover with COMPOUND‐20 for 7 days.

Conclusions and Implications

Human and rat AT, but not mouse AT, exhibited tachyphylaxis for inhibition of 11β‐HSD1 activity after repeat dosing. Translation of observed efficacy in murine disease models to human for 11β‐HSD1 inhibitors may be misleading. Investigators of the effects of 11β‐HSD1 inhibitors should confirm that desired levels of enzyme inhibition in AT can be maintained over time after repeat dosing and not rely on results following a single dose.

Abbreviations

11β‐HSD1

11β‐hydroxysteroid dehydrogenase type I

PK/PD

pharmacokinetic‐pharmacodynamic

AT

adipose tissue

DIO

diet induced obese

IHC

International Conference on Harmonisation

GCP

Good Clinical Practice

b.i.d.

twice daily

u.i.d.

once daily

HPMC

hydroxypropylmethylcellulose

IC₇₀

concentration that delivers 70% of the maximum effect

IC₉₀

concentration that delivers 90% of the maximum effect

fu

fraction unbound

C_max

maximum achieved concentration

C_min

minimum or trough concentration

E₀

baseline

E_max

maximum effect

ANCOVA

analysis of covariance