Understanding of the Viscoelastic Response of the Human Corneal Stroma Induced by Riboflavin/UV-A Cross-Linking at the Nano Level

Purpose

To investigate the viscoelastic changes of the human cornea induced by riboflavin/UV-A cross-linking using Atomic Force Microscopy (AFM) at the nano level.

Methods

Seven eye bank donor corneas were investigated, after gently removing the epithelium, using a commercial AFM in the force spectroscopy mode. Silicon cantilevers with tip radius of 10 nm and spring elastic constants between 26- and 86-N/m were used to probe the viscoelastic properties of the anterior stroma up to 3 µm indentation depth. Five specimens were tested before and after riboflavin/UV-A cross-linking; the other two specimens were chemically cross-linked using glutaraldehyde 2.5% solution and used as controls. The Young’s modulus (E) and the hysteresis (H) of the corneal stroma were quantified as a function of the application load and scan rate.

Results

The Young’s modulus increased by a mean of 1.1-1.5 times after riboflavin/UV-A cross-linking (P<0.05). A higher increase of E, by a mean of 1.5-2.6 times, was found in chemically cross-linked specimens using glutaraldehyde 2.5% (P<0.05). The hysteresis decreased, by a mean of 0.9-1.5 times, in all specimens after riboflavin/UV-A cross-linking (P<0.05). A substantial decrease of H, ranging between 2.6 and 3.5 times with respect to baseline values, was observed in glutaraldehyde-treated corneas (P<0.05).

Conclusions

The present study provides the first evidence that riboflavin/UV-A cross-linking induces changes of the viscoelastic properties of the cornea at the scale of stromal molecular interactions.     

The Effect of Riboflavin/UVA Collagen Cross-linking Therapy on the Structure and Hydrodynamic Behaviour of the Ungulate and Rabbit Corneal Stroma

Purpose

To examine the effect of riboflavin/UVA corneal crosslinking on stromal ultrastructure and hydrodynamic behaviour.

Methods

One hundred and seventeen enucleated ungulate eyes (112 pig and 5 sheep) and 3 pairs of rabbit eyes, with corneal epithelium removed, were divided into four treatment groups: Group 1 (28 pig, 2 sheep and 3 rabbits) were untreated; Group 2 (24 pig) were exposed to UVA light (3.04 mW/cm²) for 30 minutes and Group 3 (29 pig) and Group 4 (31 pig, 3 sheep and 3 rabbits) had riboflavin eye drops applied to the corneal surface every 5 minutes for 35 minutes. Five minutes after the initial riboflavin instillation, the corneas in Group 4 experienced a 30 minute exposure to UVA light (3.04 mW/cm²). X-ray scattering was used to obtain measurements of collagen interfibrillar spacing, spatial order, fibril diameter, D-periodicity and intermolecular spacing throughout the whole tissue thickness and as a function of tissue depth in the treated and untreated corneas. The effect of each treatment on the hydrodynamic behaviour of the cornea (its ability to swell in saline solution) and its resistance to enzymatic digestion were assessed using in vitro laboratory techniques.

Results

Corneal thickness decreased significantly following riboflavin application (p<0.01) and also to a lesser extent after UVA exposure (p<0.05). With the exception of the spatial order factor, which was higher in Group 4 than Group 1 (p<0.01), all other measured collagen parameters were unaltered by cross-linking, even within the most anterior 300 microns of the cornea. The cross-linking treatment had no effect on the hydrodynamic behaviour of the cornea but did cause a significant increase in its resistance to enzymatic digestion.

Conclusions

It seems likely that cross-links formed during riboflavin/UVA therapy occur predominantly at the collagen fibril surface and in the protein network surrounding the collagen.     

Riboflavin/UVA collagen cross-linking-induced changes in normal and keratoconus corneal stroma

Purpose

To determine the effect of Ultraviolet-A collagen cross-linking with hypo-osmolar and iso-osmolar riboflavin solutions on stromal collagen ultrastructure in normal and keratoconus ex vivo human corneas.

Methods

Using small-angle X-ray scattering, measurements of collagen D-periodicity, fibril diameter and interfibrillar spacing were made at 1 mm intervals across six normal post-mortem corneas (two above physiological hydration (swollen) and four below (unswollen)) and two post-transplant keratoconus corneal buttons (one swollen; one unswollen), before and after hypo-osmolar cross-linking. The same parameters were measured in three other unswollen normal corneas before and after iso-osmolar cross-linking and in three pairs of swollen normal corneas, in which only the left was cross-linked (with iso-osmolar riboflavin).

Results

Hypo-osmolar cross-linking resulted in an increase in corneal hydration in all corneas. In the keratoconus corneas and unswollen normal corneas, this was accompanied by an increase in collagen interfibrillar spacing (p<0.001); an increase in fibril diameter was also seen in two out of four unswollen normal corneas and one unswollen keratoconus cornea (p<0.001). Iso-osmolar cross-linking resulted in a decrease in tissue hydration in the swollen normal corneas only. Although there was no consistent treatment-induced change in hydration in the unswollen normal samples, iso-osmolar cross-linking of these corneas did result in a compaction of collagen fibrils and a reduced fibril diameter (p<0.001); these changes were not seen in the swollen normal corneas. Collagen D-periodicity was not affected by either treatment.

Conclusion

The observed structural changes following Ultraviolet-A cross-linking with hypo-osmolar or iso-osmolar riboflavin solutions are more likely a consequence of treatment-induced changes in tissue hydration rather than cross-linking.     

One-Step Transepithelial Topography-Guided Ablation in the Treatment of Myopic Astigmatism

Purpose

To evaluate one-step topography-guided transepithelial ablation in the treatment of low to moderate myopic astigmatism using a 1KHz excimer laser.

Methods

Retrospective study of 117 consecutive eyes available for evaluation 12 months after surgery. Pre- and post-operative visual and refractive data as well as post-operative pain and haze were analyzed. A novel technique integrating custom refractive- and epithelial- ablation in a single uninterrupted procedure was used.

Results

The mean pre-operative spherical equivalent (SE) and the mean cylinder were: –3.22 diopters (D) ±1.54 (SD) (range –0.63 to –7.25 D) and –0.77 D ±0.65 (range 0 to –4.50 D), respectively. At 12 months after surgery: no eyes lost ≥2 lines of corrected distant visual acuity (CDVA). Safety and efficacy indexes were 1.27 and 1.09, respectively. Uncorrected distant visual acuity (UDVA) was ≥20/20 in 96.6% of the eyes. Manifest refraction spherical equivalent was within ±0.5 D of the desired refraction in 93.2% of the eyes. Average root mean square (RMS) wavefront error measured at central 6 mm, increased from 0.38 pre-operatively to 0.47 µm post-operatively. Refractive stability was achieved and sustained 1 month after surgery. No visually significant haze was registered during the observation period. Post-operative pain was reported in 4.5% of patients.

Conclusions

One-step transepithelial topography-guided treatment for low to moderate myopia and astigmatism performed with a 1 KHz laser, provided safe, effective, predictable and stable results with low pain and no visually significant haze.     

Analysis of the Viscoelastic Properties of the Human Cornea Using Scheimpflug Imaging in Inflation Experiment of Eye Globes

Purpose

To demonstrate a Scheimpflug-based imaging procedure for investigating the depth- and time-dependent strain response of the human cornea to inflation testing of whole eye globes.

Methods

Six specimens, three of which with intact corneal epithelium, were mounted in a customized apparatus within a humidity and temperature-monitored wet chamber. Each specimen was subjected to two mechanical tests in order to measure corneal strain resulting from application of cyclic (cyclic regimen) and constant (creep regimen) stress by changing the intra-ocular pressure (IOP) within physiological ranges (18–42 mmHg). Corneal shape changes were analyzed as a function of IOP and both corneal stress-strain curves and creep curves were generated.

Results

The procedure was highly accurate and repeatable. Upon cyclic stress application, a biomechanical corneal elasticity gradient was found in the front-back direction. The average Young''s modulus of the anterior cornea ranged between 2.28±0.87 MPa and 3.30±0.90 MPa in specimens with and without intact epithelium (P = 0.05) respectively. The Young''s modulus of the posterior cornea was on average 0.21±0.09 MPa and 0.17±0.06 MPa (P>0.05) respectively. The time-dependent strain response of the cornea to creep testing was quantified by fitting data to a modified Zener model for extracting both the relaxation time and compliance function.

Conclusion

Cyclic and creep mechanical tests are valuable for investigating the strain response of the intact human cornea within physiological IOP ranges, providing meaningful results that can be translated to clinic. The presence of epithelium influences the results of anterior corneal shape changes when monitoring deformation via Scheimpflug imaging in inflation experiments of whole eye globes.     

Assessment of Choroidal Thickness in Healthy and Glaucomatous Eyes Using Swept Source Optical Coherence Tomography

Purpose

To evaluate choroidal thickness (CT) in healthy and glaucomatous eyes using Swept Source Optical Coherence Tomography (SS-OCT).

Methods

A cross-sectional observational study of 216 eyes of 140 subjects with glaucoma and 106 eyes of 67 healthy subjects enrolled in the Diagnostic Innovations in Glaucoma Study. CT was assessed from wide-field (12×9 mm) SS-OCT scans. The association between CT and potential confounding variables including age, gender, axial length, intraocular pressure, central corneal thickness and ocular perfusion pressure was examined using univariable and multivariable regression analyses.

Results

Overall CT was thinner in glaucomatous eyes with a mean (± standard deviation) of 157.7±48.5 µm in glaucoma compared to 179.9±36.1 µm in healthy eyes (P<0.001). The choroid was thinner in both the peripapillary and macular regions in glaucoma compared to controls. Mean peripapillary CT was 154.1±44.1 µm and 134.0±56.9 µm (P<0.001) and macular CT 199.3±46.1 µm and 176.2±57.5 µm (P<0.001) for healthy and glaucomatous eyes respectively. However, older age (P<0.001) and longer axial length (P<0.001) were also associated with thinner choroid and when differences in age and axial length between glaucomatous and healthy subjects were accounted for, glaucoma was not significantly associated with CT. There was also no association between glaucoma severity and CT.

Conclusions

Glaucoma was not associated with CT measured using SS-OCT; however, older age and longer axial length were associated with thinner choroid so should be considered when interpreting CT measurements.     

Anatomical Characteristics of Pulmonary Veins for the Prediction of Postoperative Recurrence after Radiofrequency Catheter Ablation of Atrial Fibrillation

Background

The relationship between focal pulmonary vein potential and atrial fibrillation (AF) has been confirmed. Pulmonary vein (PV) isolation and circumferential pulmonary vein ablation have been the most commonly used procedures of radiofrequency ablation. However, few studies have investigated the relationship between anatomical characteristics of PV and AF recurrences after radiofrequency ablation.

Methodology

For 267 AF patients treated by radiofrequency catheter ablation, the anatomic structure characteristics of pulmonary veins were assessed by multi-slice spiral computed tomography while the values of left atrial diameter (LAD) were measured with transesophageal ultrasonic cardiogram. After radiofrequency catheter ablation, postoperative recurrence was evaluated during a 10-month term follow-up.

Principal Findings

During follow-up, postoperative recurrence occurred in 44 patients. The mean diameters of LAD, left superior PV, right superior PV, all left PV, and all superior PV were significantly larger in patients with postoperative recurrence (Recurrence vs. Non-recurrence group; 43.9 ± 6.4 mm vs. 40.7 ± 5.6 mm; 18.4 ± 2.1 mm vs. 17.1 ± 3.1 mm; 18.2 ± 2.8 mm vs. 17.2 mm ± 3.9 mm; 16.4 ± 1.5 mm vs. 15.6 ± 2.5 mm; 18.3 ± 2.1 mm vs. 17.1 ± 3.0 mm; respectively; all P < 0.05). Multivariable survival analysis showed that the type and the course of AF, LAD, and the diameters of all superior PV were the independent risk factors for the postoperative recurrence after radiofrequency catheter ablation.

Conclusions

The enlargements of all superior PV and LAD, long course of diseases, and persistent AF were the independent risk factors for the postoperative recurrence after radiofrequency catheter ablation.     

Evaluation of the Shape Symmetry of Bilateral Normal Corneas in a Chinese Population

Purpose

To investigate the bilateral symmetry of the global corneal topography in normal corneas with a wide range of curvature, astigmatism and thickness values

Design

Cross-Sectional Study

Methods

Topography images were recorded for the anterior and posterior surfaces of 342 participants using a Pentacam. Elevation data were fitted to a general quadratic model that considered both translational and rotational displacements. Comparisons between fellow corneas of estimates of corneal shape parameters (elevation, radius in two main directions, R_x and R_y, and corresponding shape factors, Q_x and Q_y) and corneal position parameters (translational displacements: x₀, y₀ and z₀, and rotational displacements: α, β and γ) were statistically analyzed.

Results

The general quadratic model provided average RMS of fit errors with the topography data of 1.7±0.6 µm and 5.7±1.3 µm in anterior and posterior corneal surfaces. The comparisons showed highly significant bilateral correlations with the differences between fellow corneas in R_x, R_y, Q_x and Q_y of anterior and posterior surfaces remaining insignificantly different from zero. Bilateral differences in elevation measurements at randomly-selected points in both corneal central and peripheral areas indicated strong mirror symmetry between fellow corneas. The mean geometric center (x₀, y₀, z₀) of both right and left corneas was located on the temporal side and inferior-temporal side of the apex in anterior and posterior topography map, respectively. Rotational displacement angle α along X axis had similar distributions in bilateral corneas, while rotation angle β along Y axis showed both eyes tilting towards the nasal side. Further, rotation angle γ along Z axis, which is related to corneal astigmatism, showed clear mirror symmetry.

Conclusions

Analysis of corneal topography demonstrated strong and statistically-significant mirror symmetry between bilateral corneas. This characteristic could help in detection of pathological abnormalities, disease diagnosis, measurement validation and surgery planning.     

Ultrastructural study of collagen fibrils,proteoglycans and lamellae of the cornea treated with iontophoresis – UVA cross-linking and hypotonic riboflavin solution

To investigate the effects of iontophoresis–ultraviolet A (UVA) cross-linking (CXL) with hypotonic riboflavin solution on the ultrastructural changes in the lamellae, collagen fibrils (CFs), and proteoglycans (PGs) in the central and peripheral stroma of the human corneal buttons. The iontophoresis method was used for the trans-epithelial application of hypotonic riboflavin in ex vivo corneal culture for 5 min. The corneas were irradiated using three methods: Group 1 (G1), a UVA irradiance of 3 mW/cm² for 30 min; Group 2 (G2), a UVA irradiance of 10 mW/cm² for 9 min; Group 3 (G3), without UVA irradiation. Three untreated corneas were used as controls (G0). After the CXL procedure, the corneas were processed for electron microscopy. The CF diameter and PGs in each sample were analyzed using the iTEM program. The keratocyte organelles and stromal architecture in the peripheral cornea were better preserved than those in the central cornea. In G1 and G2, the mean CF diameter in the peripheral cornea was significantly higher than that in the central cornea. In G3, the CF diameter in the central cornea was significantly larger than that in the peripheral cornea. Furthermore, differences in PG area size were observed between the central and peripheral corneas in all groups. Riboflavin + UVA application at 3 mW/cm² for 30 min and 10 mW/cm² for 9 min was a suitable method of CXL; however, 3 mW/cm² for 30 min improved the organization and size of the collagen fibrils. CXL treatment applied at the periphery was more effective than that applied at the center.Keyword: Collagen fibrils, Proteoglycans, Cornea, Iontophoresis, Ultraviolet A     

De Novo Lipogenesis and Cholesterol Synthesis in Humans with Long-Standing Type 1 Diabetes Are Comparable to Non-Diabetic Individuals

Background

Synthesis of lipid species, including fatty acids (FA) and cholesterol, can contribute to pathological disease. The purpose of this study was to investigate FA and cholesterol synthesis in individuals with type 1 diabetes, a group at elevated risk for vascular disease, using stable isotope analysis.

Methods

Individuals with type 1 diabetes (n = 9) and age-, sex-, and BMI-matched non-diabetic subjects (n = 9) were recruited. On testing day, meals were provided to standardize food intake and elicit typical feeding responses. Blood samples were analyzed at fasting (0 and 24 h) and postprandial (2, 4, 6, and 8 hours after breakfast) time points. FA was isolated from VLDL to estimate hepatic FA synthesis, whereas free cholesterol (FC) and cholesteryl ester (CE) was isolated from plasma and VLDL to estimate whole-body and hepatic cholesterol synthesis, respectively. Lipid synthesis was measured using deuterium incorporation and isotope ratio mass spectrometry.

Results

Fasting total hepatic lipogenesis (3.91±0.90% vs. 5.30±1.22%; P = 0.41) was not significantly different between diabetic and control groups, respectively, nor was synthesis of myristic (28.60±4.90% vs. 26.66±4.57%; P = 0.76), palmitic (12.52±2.75% vs. 13.71±2.64%; P = 0.65), palmitoleic (3.86±0.91% vs. 4.80±1.22%; P = 0.65), stearic (5.55±1.04% vs. 6.96±0.97%; P = 0.29), and oleic acid (1.45±0.28% vs. 2.10±0.51%; P = 0.21). Postprandial lipogenesis was also not different between groups (P = 0.38). Similarly, fasting synthesis of whole-body FC (8.2±1.3% vs. 7.3±0.8%/day; P = 0.88) and CE (1.9±0.4% vs. 2.0±0.3%/day; P = 0.96) and hepatic FC (8.2±2.0% vs. 8.1±0.8%/day; P = 0.72) was not significantly different between diabetic and control subjects.

Conclusions

Despite long-standing disease, lipogenesis and cholesterol synthesis was not different in individuals with type 1 diabetes compared to healthy non-diabetic humans.     

The Morphology of Corneal Cap and Its Relation to Refractive Outcomes in Femtosecond Laser Small Incision Lenticule Extraction (SMILE) with Anterior Segment Optical Coherence Tomography Observation

Purpose

To investigate the morphology of corneal caps in femtosecond laser small incision lenticule extraction (SMILE) and its relation to the refractive outcomes.

Methods

A prospective study of fifty-four corneal caps created with VisuMax femtosecond laser were examined using an Fourier-domain optical coherence tomography at 1 day, 1 week, 1 month and 6 months after SMILE. The cap thickness at nine points on each of the four meridians (0°, 45°, 90°, 135°) and the diameter were measured. Cap morphology, changes over time and its correlation with refractive outcomes were assessed.

Results

The mean achieved central cap thickness were (108.74±5.06) µm at 6 months and (107.32±4.81 ) µm at 1 month postoperatively, significantly thinner than that at 1 day (110.81±7.95) µm and 1 week (109.58±7.48 ) µm (P<0.05). The mean diameter on 0° meridian was (7.61±0.07) mm, significantly larger than that on 90° meridian (7.57±0.06) mm (P = 0.001). Cap morphology showed good regularity, except that the differences of points in two pairs were significant at 1 day postoperatively. The uniformity was consistent over time and the central cap thickness was thinner than those in the paracentral and peripheral areas. The refractive outcomes stabilized within 1 month. Uncorrected distance visual acuity (UDVA) was correlated to the central cap thickness at 1 day and 1 week (both r_s = 0.33, p<0.05). The uniformity index was correlated with UDVA (r_s = 0.34, p<0.05) and corrected distance visual acuity (r_s = 0.32, p<0.05) at 1 week postoperatively.

Conclusions

Corneal caps of SMILE are predictable with good reproducibility, regularity and uniformity. Cap morphology might have a mild effect on refractive outcomes in the early stage. Further study should focus on the impact on the visual quality.     

Evaluation of the Quality of Guidelines for Myasthenia Gravis with the AGREE II Instrument

Background

Clinical practice guidelines (CPGs) are systematically developed statements to assist practitioners in making decisions about appropriate healthcare in specific clinical circumstances. The methodological quality of CPGs for myasthenia gravis (MG) are unclear.

Objective

To critically evaluate the methodological quality of CPGs for MG using AGREE II instrument.

Method

A systematical search strategy on PubMed, EMBASE, DynaMed, the National Guideline Clearinghouse (NGC) and the Chinese Biomedical Literature database (CBM) was performed on September 20th 2013. All guidelines related to MG were evaluated with AGREE II. The software used for analysis was SPSS 17.0.

Results

A total of 15 CPGs for MG met the inclusion criteria (12 CPGs in English, 3 CPGs in Chinese). The overall agreement among reviews was moderate or high (ICC >0.70). The mean scores (mean ± SD) for al six domains were presented as follows: scope and purpose (60.93% ±16.62%), stakeholder involvement (40.93% ±20.04%), rigor of development (37.22% ±30.46%), clarity of presentation (64.26% ±16.36%), applicability (28.19% ±20.56%) and editorial independence (27.78% ±28.28%). Compared with non-evidence-based CPGs, evidence-based CPGs had statistically significant higher quality scores for all AGREE II domains (P<0.05). All domain scores appear slightly higher for CPGs published after AGREE II instrument development and validation (P>0.05). The quality scores of CPGs developed by NGC/AAN were higher than the quality scores of CPGs developed by other organizations for all domains. The difference was statistically significant for all domains with the exception of clarity of presentation (P = 0.07).

Conclusions

The qualities of CPGs on MG were generally acceptable with several flaws. The AGREE II instrument should be adopted by guideline developers, particularly in China.     

Hemodynamic Effects of Stent Struts versus Straightening of Vessels in Stent-Assisted Coil Embolization for Sidewall Cerebral Aneurysms

Background

Recent clinical studies have shown that recanalization rates are lower in stent-assisted coil embolization than in coiling alone in the treatment of cerebral aneurysms.

Objective

This study aimed to assess and compare the hemodynamic effect of stent struts and straightening of vessels by stent placement on reducing flow velocity in sidewall aneurysms, with the goal of reducing recanalization rates.

Methods

We evaluated 16 sidewall aneurysms treated with Enterprise stents. We performed computational fluid dynamics simulations using patient-specific geometries before and after treatment, with or without stent struts.

Results

Stent placement straightened vessels by a mean (±standard deviation) of 12.9°±13.1° 6 months after treatment. Placement of stent struts in the initial vessel geometries reduced flow velocity in aneurysms by 23.1%±6.3%. Straightening of vessels without stent struts reduced flow velocity by 9.6%±12.6%. Stent struts had significantly stronger effects on reducing flow velocity than straightening (P = 0.004, Wilcoxon test). Deviation of the effects was larger by straightening than by stent struts (P = 0.01, F-test). The combination of stent struts and straightening reduced flow velocity by 32.6%±12.2%. There was a trend that larger inflow angles produced a larger reduction in flow velocity by straightening of vessels (P = 0.16).

Conclusion

In sidewall aneurysms, stent struts have stronger effects (approximately 2 times) on reduction in flow velocity than straightening of vessels. Hemodynamic effects by straightening vary in each case and can be predicted by inflow angles of pre-operative vessel geometry. These results may be useful to design a treatment strategy for reducing recanalization rates.     

Comparison of Sum Absolute QRST Integral,and Temporal Variability in Depolarization and Repolarization,Measured by Dynamic Vectorcardiography Approach,in Healthy Men and Women

Background

Recently we showed the predictive value of sum absolute QRST integral (SAI QRST) and repolarization lability for risk stratification of sudden cardiac death (SCD) in heart failure patients. The goal of this study was to compare SAI QRST and metrics of depolarization and repolarization variability in healthy men and women.

Methods

Orthogonal ECGs were recorded at rest for 10 minutes in 160 healthy men and women (mean age 39.6±14.6, 80 men). Mean spatial TT′ angle, and normalized variances of T loop area, of spatial T vector amplitude, of QT interval and Tpeak-Tend area were measured for assessment of repolarization lability. Normalized variances of spatial QRS vector and QRS loop area characterized variability of depolarization. In addition, variability indices (VI) were calculated to adjust for normalized heart rate variance. SAI QRST was measured as the averaged arithmetic sum of areas under the QRST curve.

Results

Men were characterized by shorter QTc (430.3±21.7 vs. 444.7±22.2 ms; P<0.0001) and larger SAI QRST (282.1±66.7 vs.204.9±58.5 mV*ms; P<0.0001). Repolarization lability negatively correlated with spatial T vector amplitude. Adjusted by normalized heart rate variance, QT variability index was significantly higher in women than in men (−1.54±0.38 vs. −1.70±0.33; P = 0.017). However, in multivariate logistic regression after adjustment for body surface area, QTc, and spatial T vector amplitude, healthy men had 1.5–3 fold higher probability of having larger repolarization lability, as compared to healthy women (T vector amplitude variability index odds ratio 3.88(95%CI 1.4–11.1; P = 0.012).

Conclusions

Healthy men more likely than women have larger repolarization lability.     

Antenatal Dexamethasone after Asphyxia Increases Neural Injury in Preterm Fetal Sheep

Background and Purpose

Maternal glucocorticoid treatment for threatened premature delivery dramatically improves neonatal survival and short-term morbidity; however, its effects on neurodevelopmental outcome are variable. We investigated the effect of maternal glucocorticoid exposure after acute asphyxia on injury in the preterm brain.

Methods

Chronically instrumented singleton fetal sheep at 0.7 of gestation received asphyxia induced by complete umbilical cord occlusion for 25 minutes. 15 minutes after release of occlusion, ewes received a 3 ml i.m. injection of either dexamethasone (12 mg, n = 10) or saline (n = 10). Sheep were killed after 7 days recovery; survival of neurons in the hippocampus and basal ganglia, and oligodendrocytes in periventricular white matter were assessed using an unbiased stereological approach.

Results

Maternal dexamethasone after asphyxia was associated with more severe loss of neurons in the hippocampus (CA3 regions, 290±76 vs 484±98 neurons/mm², mean±SEM, P<0.05) and basal ganglia (putamen, 538±112 vs 814±34 neurons/mm², P<0.05) compared to asphyxia-saline, and with greater loss of both total (913±77 vs 1201±75/mm², P<0.05) and immature/mature myelinating oligodendrocytes in periventricular white matter (66±8 vs 114±12/mm², P<0.05, vs sham controls 165±10/mm², P<0.001). This was associated with transient hyperglycemia (peak 3.5±0.2 vs. 1.4±0.2 mmol/L at 6 h, P<0.05) and reduced suppression of EEG power in the first 24 h after occlusion (maximum −1.5±1.2 dB vs. −5.0±1.4 dB in saline controls, P<0.01), but later onset and fewer overt seizures.

Conclusions

In preterm fetal sheep, exposure to maternal dexamethasone during recovery from asphyxia exacerbated brain damage.     

Sodium Bicarbonate Treatment during Transient or Sustained Lactic Acidemia in Normoxic and Normotensive Rats

Introduction

Lactic acidosis is a frequent cause of poor outcome in the intensive care settings. We set up an experimental model of lactic acid infusion in normoxic and normotensive rats to investigate the systemic effects of lactic acidemia per se without the confounding factor of an underlying organic cause of acidosis.

Methodology

Sprague Dawley rats underwent a primed endovenous infusion of L(+) lactic acid during general anesthesia. Normoxic and normotensive animals were then randomized to the following study groups (n = 8 per group): S) sustained infusion of lactic acid, S+B) sustained infusion+sodium bicarbonate, T) transient infusion, T+B transient infusion+sodium bicarbonate. Hemodynamic, respiratory and acid-base parameters were measured over time. Lactate pharmacokinetics and muscle phosphofructokinase enzyme''s activity were also measured.

Principal Findings

Following lactic acid infusion blood lactate rose (P<0.05), pH (P<0.05) and strong ion difference (P<0.05) drop. Some rats developed hemodynamic instability during the primed infusion of lactic acid. In the normoxic and normotensive animals bicarbonate treatment normalized pH during sustained infusion of lactic acid (from 7.22±0.02 to 7.36±0.04, P<0.05) while overshoot to alkalemic values when the infusion was transient (from 7.24±0.01 to 7.53±0.03, P<0.05). When acid load was interrupted bicarbonate infusion affected lactate wash-out kinetics (P<0.05) so that blood lactate was higher (2.9±1 mmol/l vs. 1.0±0.2, P<0.05, group T vs. T+B respectively). The activity of phosphofructokinase enzyme was correlated with blood pH (R2 = 0.475, P<0.05).

Conclusions

pH decreased with acid infusion and rose with bicarbonate administration but the effects of bicarbonate infusion on pH differed under a persistent or transient acid load. Alkalization affected the rate of lactate disposal during the transient acid load.     

Compromised Dynamic Cerebral Autoregulation in Patients with a Right-to-Left Shunt: A Potential Mechanism of Migraine and Cryptogenic Stroke

Background and Purpose

The relationship between right-to-left shunts (RLS) and migraine and cryptogenic stroke is not well understood. In this study, we investigated whether RLS are associated with impairment of dynamic cerebral autoregulation (dCA), which may play a role in migraine and cryptogenic stroke.

Methods

Sixty-six migraineurs were enrolled in the study, including 36 non-RLS patients and 30 RLS patients. Non-invasive continuous cerebral blood flow velocity and arterial blood pressure were recorded simultaneously from each patient by using transcranial Doppler and servo-controlled plethysmograph, respectively. Transfer function analysis was applied to derive autoregulatory parameters of gain, phase difference (PD), and autoregulation index.

Results

The PD in migraineurs with RLS was 50.6±22.9 degrees, which was significantly lower than that observed in the non-RLS group (67.2±18.2 degrees, P<0.001). The PD in the large RLS group (45.4±22.6 degrees) was significantly lower than that of the small RLS group (64.9±17.1 degrees, P<0.01) and non-RLS group (P<0.001); however, the PD in the small RLS group was similar to that of the non-RLS group. The PD in the permanent group (48.8±19.9 degrees) was similar to that of the latent group (52.6±26.1 degrees), and both were significantly lower than that of the non-RLS group (P<0.05). The autoregulation index results were similar to the PD findings.

Conclusions

dCA is impaired in migraineurs with large RLS, and this may represent a potential mechanism linking RLS, migraine, and cryptogenic stroke.     

Metabolic Imaging of Human Kidney Triglyceride Content: Reproducibility of Proton Magnetic Resonance Spectroscopy

Objective

To assess the feasibility of renal proton magnetic resonance spectroscopy for quantification of triglyceride content and to compare spectral quality and reproducibility without and with respiratory motion compensation in vivo.

Materials and Methods

The Institutional Review Board of our institution approved the study protocol, and written informed consent was obtained. After technical optimization, a total of 20 healthy volunteers underwent renal proton magnetic resonance spectroscopy of the renal cortex both without and with respiratory motion compensation and volume tracking. After the first session the subjects were repositioned and the protocol was repeated to assess reproducibility. Spectral quality (linewidth of the water signal) and triglyceride content were quantified. Bland-Altman analyses and a test by Pitman were performed.

Results

Linewidth changed from 11.5±0.4 Hz to 10.7±0.4 Hz (all data pooled, p<0.05), without and with respiratory motion compensation respectively. Mean % triglyceride content in the first and second session without respiratory motion compensation were respectively 0.58±0.12% and 0.51±0.14% (P = NS). Mean % triglyceride content in the first and second session with respiratory motion compensation were respectively 0.44±0.10% and 0.43±0.10% (P = NS between sessions and P = NS compared to measurements with respiratory motion compensation). Bland-Altman analyses showed narrower limits of agreement and a significant difference in the correlated variances (correlation of −0.59, P<0.05).

Conclusion

Metabolic imaging of the human kidney using renal proton magnetic resonance spectroscopy is a feasible tool to assess cortical triglyceride content in humans in vivo and the use of respiratory motion compensation significantly improves spectral quality and reproducibility. Therefore, respiratory motion compensation seems a necessity for metabolic imaging of renal triglyceride content in vivo.     

Novel Cell-Free Strategy for Therapeutic Angiogenesis: In Vitro Generated Conditioned Medium Can Replace Progenitor Cell Transplantation

Background

Current evidence suggests that endothelial progenitor cells (EPC) contribute to ischemic tissue repair by both secretion of paracrine factors and incorporation into developing vessels. We tested the hypothesis that cell-free administration of paracrine factors secreted by cultured EPC may achieve an angiogenic effect equivalent to cell therapy.

Methodology/Principal Findings

EPC-derived conditioned medium (EPC-CM) was obtained from culture expanded EPC subjected to 72 hours of hypoxia. In vitro, EPC-CM significantly inhibited apoptosis of mature endothelial cells and promoted angiogenesis in a rat aortic ring assay. The therapeutic potential of EPC-CM as compared to EPC transplantation was evaluated in a rat model of chronic hindlimb ischemia. Serial intramuscular injections of EPC-CM and EPC both significantly increased hindlimb blood flow assessed by laser Doppler (81.2±2.9% and 83.7±3.0% vs. 53.5±2.4% of normal, P<0.01) and improved muscle performance. A significantly increased capillary density (1.62±0.03 and 1.68±0.05/muscle fiber, P<0.05), enhanced vascular maturation (8.6±0.3 and 8.1±0.4/HPF, P<0.05) and muscle viability corroborated the findings of improved hindlimb perfusion and muscle function. Furthermore, EPC-CM transplantation stimulated the mobilization of bone marrow (BM)-derived EPC compared to control (678.7±44.1 vs. 340.0±29.1 CD34⁺/CD45⁻ cells/1×10⁵ mononuclear cells, P<0.05) and their recruitment to the ischemic muscles (5.9±0.7 vs. 2.6±0.4 CD34⁺ cells/HPF, P<0.001) 3 days after the last injection.

Conclusions/Significance

Intramuscular injection of EPC-CM is as effective as cell transplantation for promoting tissue revascularization and functional recovery. Owing to the technical and practical limitations of cell therapy, cell free conditioned media may represent a potent alternative for therapeutic angiogenesis in ischemic cardiovascular diseases.     

The Association of Homocysteine with Metabolic Syndrome in a Community-Dwelling Population: Homocysteine Might Be Concomitant with Metabolic Syndrome

Objective

Elevated plasma total homocysteine (tHcy) and metabolic syndrome (MetS) are both associated with cardiovascular disease, but the association between tHcy and MetS is not well characterized. The aim of this study was to determine the relationship between tHcy and MetS.

Methods

To further estimate the time-dependent association of tHcy and MetS, we analyzed the tHcy level and MetS in 1499 subjects from a 4.8-year longitudinal study in Beijing, People’s Republic of China.

Results

In multiple linear regression analysis, baseline tHcy levels associated with age, BMI, SBP, DBP, LDL-C and Cr independently over 4.8-years follow-up; age, BMI, SBP, DBP and Cr were found to be associated with tHcy levels independently at the end of follow-up. Logistic regression analysis showed that there was no association between the baseline tHcy level and MetS over the 4.8-year follow-up (odds ratio (OR), 1.32; 95% confidence interval (CI), 0.79–2.19; P = 0.282); rather, there was an association only with hypertension as a MetS component (OR, 1.53; 95% CI, 1.06–2.21; P = 0.024). tHcy levels were associated with MetS at both cross-sectional baseline (OR, 1.38; 95% CI, 1.02–1.88; P = 0.038) and cross-sectional follow-up (OR, 1.60; 95% CI, 1.02–2.50; P = 0.041). The tHcy levels of MetS subjects were higher than those of non-MetS subjects at both cross-sectional baseline (19.35±7.92 µmol/L vs. 17.45±6.70 µmol/L, respectively; P = 0.001) and cross-sectional follow-up (18.95±7.15 µmol/L vs. 17.11±5.98 µmol/L, respectively; P = 0.02).

Conclusion

The tHcy level was not predictive of the incidence of MetS; however, it may be a risk factor for hypertension as a MetS component. Furthermore, tHcy levels were associated with MetS at cross-sectional baseline and follow-up, which suggests that a higher level of tHcy might be concomitant with MetS.