Cell Therapy for Parkinson's Disease: A Translational Approach to Assess the Role of Local and Systemic Immunosuppression.

Neural transplantation is a promising therapeutic approach for neurodegenerative diseases; however, many patients receiving intracerebral fetal allografts exhibit signs of immunization to donor antigens that could compromise the graft. In this context, we intracerebrally transplanted mesencephalic pig xenografts into primates to identify a suitable strategy to enable long-term cell survival, maturation, and differentiation. Parkinsonian primates received WT or CTLA4-Ig transgenic porcine xenografts and different durations of peripheral immunosuppression to test whether systemic plus graft-mediated local immunosuppression might avoid rejection. A striking recovery of spontaneous locomotion was observed in primates receiving systemic plus local immunosuppression for 6 mo. Recovery was associated with restoration of dopaminergic activity detected both by positron emission tomography imaging and histological examination. Local infiltration by T cells and CD80/86+ microglial cells expressing indoleamine 2,3-dioxigenase were observed only in CTLA4-Ig recipients. Results suggest that in this primate neurotransplantation model, peripheral immunosuppression is indispensable to achieve the long-term survival of porcine neuronal xenografts that is required to study the beneficial immunomodulatory effect of local blockade of T cell costimulation.

Measures of atherosclerotic burden are associated with clinically manifest cardiovascular disease in type 2 diabetes: a European cross-sectional study.

BACKGROUND: There is a need to develop and validate surrogate markers of cardiovascular disease (CVD) in subjects with diabetes. The macrovascular changes associated with diabetes include aggravated atherosclerosis, increased arterial stiffness and endothelial dysfunction. The aim of this study was to determine which of these factors is most strongly associated with clinically manifest cardiovascular events. METHODS: Vascular changes were measured in a cohort of 458 subjects with type 2 diabetes (T2D) and CVD (myocardial infarction, stroke or lower extremity arterial disease), 527 subjects with T2D but without clinically manifest CVD and 515 subjects without T2D and with or without CVD. RESULTS: Carotid intima-media thickness (IMT) and ankle-brachial pressure index were independently associated with the presence of CVD in subjects with T2D, whereas pulse wave velocity and endothelial function provided limited independent additive information. Measurement of IMT in the carotid bulb provided better discrimination of the presence of CVD in subjects with T2D than measurement of IMT in the common carotid artery. The factors most significantly associated with increased carotid IMT in T2D were age, disease duration, systolic blood pressure, impaired renal function and increased arterial stiffness, whereas there were no or weak independent associations with metabolic factors and endothelial dysfunction. CONCLUSIONS: Measures of atherosclerotic burden are associated with clinically manifest CVD in subjects with T2D. In addition, vascular changes that are not directly related to known metabolic risk factors are important in the development of both atherosclerosis and CVD in T2D. A better understanding of the mechanisms involved is crucial for enabling better identification of CVD risk in T2D.

HDL lipid composition is profoundly altered in patients with type 2 diabetes and atherosclerotic vascular disease.

BACKGROUND AND AIMS: We have previously shown that the anti-inflammatory and anti-oxidant functions of HDL are impaired in T2D patients. In this study, we examined whether HDL from T2D patients contains elevated levels of oxidized fatty acids and whether those levels correlate with cardiovascular disease (CVD). METHODS AND RESULTS: HETEs and HODEs on HDL were determined by LC-MS/MS in 40 non-diabetic controls (ND), 40 T2D without CVD (D⁺CVD⁻) and 38 T2D with known history of CVD (D⁺CVD⁺). HDL oxidant index was evaluated by a cell-free assay using dichlorofluorescein. Twenty-six randomly selected subjects from the three groups underwent coronary calcium score evaluation (CAC). Major cardiovascular risk factors were similar among the groups. HETEs and HODEs content were significantly increased in HDL from D⁺CVD⁺ when compared to D⁺CVD⁻ and ND patients. HDL oxidant index was not different among the three groups; however, it was significantly higher in patients with CAC score >100 when compared to patients with CAC score <100. CONCLUSION: Patients with D⁺CVD⁻ and D⁺CVD⁺ are characterized by a severe, graded enrichment of oxidized fatty acids on HDL. In the present study, a loss of HDL function (as estimated by the HDL oxidant index) is observed only in patients with more advanced atherosclerosis.

Systemic inhibition of nitric oxide synthesis in non-diabetic individuals produces a significant deterioration in glucose tolerance by increasing insulin clearance and inhibiting insulin secretion.

AIMS/HYPOTHESIS: Endogenous NO inhibits insulin release in isolated beta cells and insulin-degrading enzyme activity in hepatocytes, while NO release from endothelial cells has been suggested to enhance insulin action. We assessed the overall effect of systemic inhibition of endogenous NO synthesis on glucose homeostasis in humans. METHODS: Twenty-four non-diabetic volunteers underwent two hyperglycaemic (+7 mmol/l) clamps with either saline or L-NG-nitroarginine methyl ester (L-NAME, at rates of 2.5, 5, 10 and 20 µg min⁻¹ kg⁻¹) infusion. Another five volunteers underwent an OGTT with either saline or L-NAME (20 µg min⁻¹ kg⁻¹) infusion. Blood pressure and heart rate were measured to monitor NO blockade; during the OGTT, endothelial function was assessed by peripheral arterial tonometry and insulin secretion by C-peptide deconvolution and insulin secretion modelling. RESULTS: Compared with saline, L-NAME at the highest dose raised mean blood pressure (+20 ± 2 mmHg), depressed heart rate (-12 ± 2 bpm) and increased insulin clearance (+50%). First-phase insulin secretion was impaired, but insulin sensitivity (M/I index) was unchanged. During the OGTT, L-NAME raised 2 h plasma glucose by 1.8 mmol/l (p < 0.01), doubled insulin clearance and impaired beta cell glucose sensitivity while depressing endothelial function. CONCLUSIONS/INTERPRETATION: In humans, systemic NO blockade titrated to increase blood pressure and induce endothelial dysfunction does not affect insulin action but significantly impairs glucose tolerance by increasing plasma insulin clearance and depressing insulin secretion, namely first-phase and beta cell glucose sensitivity.

Salivary and urinary metabolome analysis for pre-puberty-related biomarkers identification in porcine.

Estrus synchronization is important for optimal management of gilt reproduction in pig farms. Hormonal treatments, such as synthetic progestogens, are used on a routine basis, but there is a growing demand for non-hormonal alternative breeding tools. Before puberty, gilts exhibit a 'waiting period,' related to the ovarian development and gonadotrophin secretions, during which external stimulations, such as boar exposure, could induce and synchronize first ovulation. Practical non-invasive tools for identification of this period in farms are lacking. During this period, urinary oestrone levels are high, but urine sampling is difficult in group-housed females. The aim of this work was to search for specific biomarkers of the 'waiting period' in saliva and urine. In total, nine 144- to 147-day-old Large White gilts were subjected to trans-abdominal ultrasonography three times a week for 5 weeks until puberty detection (week -5 to week -1 before puberty). Urine and saliva samples were collected for oestrone assay to detect the 'waiting period' and for metabolome analysis using 1H-nuclear magnetic resonance spectroscopy to detect potential biomarkers of the 'waiting period.' Gilts were slaughtered 7 days after puberty detection for puberty confirmation. Results were consistent with ultrasonography data for six gilts. Urine and saliva samples from these six gilts were analyzed. Urinary estrone concentration significantly increased 2 weeks before puberty detection. Metabolome analysis of urine samples allowed the identification of 78 spectral bins, among them, 42 low-molecular-weight metabolites were identified. Metabolome analysis of salivary samples allowed the identification of 59 spectral bins, among them, 23 low-molecular-weight metabolites were detected and 17 were identified. No potential biomarker was identified in urinary samples. In saliva, butyrate and 2HOvalerate, 5.79 ppm (putatively uridine), formate, malonate and propionate could be biomarker candidates to ascertain the pre-puberty period in gilt reproduction. These results confirm that non-invasive salivary samples could allow the identification of the physiological status of the gilts and presumably the optimal time for application of the boar effect. This could contribute to synchronize puberty onset and hence to develop non-hormonal breeding tools.

Ultrasonographic measures of body fatness and their relationship with plasma levels and adipose tissue expression of four adipokines in Welsh pony mares.

Obesity is responsible for metabolic dysregulations that alter fertility and induce pathologies. The objectives of the present study were to validate a reliable method for the evaluation of body fatness in mares and to associate the body fat estimation data to metabolic changes, including adipokines at the plasma and adipose tissue levels. To reach this purpose, animals were subjected to two extreme breeding conditions to study the variation of morphological, ultrasound, and physiological parameters. Twenty Welsh mares were followed up monthly from April to October before and after animals were moved outdoors to grasslands. Body weight (BW), body length (BL), height at the withers (HW), thoracic perimeter (TP), 5-point body condition score (BCS), and subcutaneous fat thickness (SFT) at the level of the shoulder, the lumbar region, and the rump, measured by ultrasonography, and plasma and adipose tissue metabolic indicators were assessed in parallel. Statistical analysis was performed using a linear mixed-effects model, whereas Pearson tests were used for the analysis of the correlations between the different parameters. Although mean BW did not increase significantly (P = 0.0940), TP (P = 0.0002) and BCS (P < 0.0001) increased during the study period. Ultrasonographic examination of subcutaneous adipose tissue showed an increase in SFT at the level of the shoulder (P < 0.0001), lumbar region (P < 0.0001), and rump (P < 0.0001). Plasma concentrations of nonesterified fatty acids (P < 0.0001), phospholipids (P < 0.0001), and cholesterol (P < 0.0001) increased significantly, whereas triglycerides (P < 0.0001) decreased significantly during the study period. Although both plasma concentrations and adipose tissue expression of leptin (P < 0.0001) and resistin (P < 0.0001) increased significantly, adiponectin (P < 0.0001) significantly decreased and visfatin remained unchanged (P = 0.8401). Expression of adipokine receptors studied showed the opposite pattern compared with their ligand. Ultrasonographic measurements of subcutaneous adipose tissue thickness at the shoulder, lumbar region, and rump are relevant indicators of fatness related with adipokine plasma concentrations and expression of adipokine-related receptors in adipose tissue, and particularly highlight seasonal effects.

Development of OPS vitrified pig blastocysts: effects of size of the collected blastocysts, cryoprotectant concentration used for vitrification and number of blastocysts transferred.

Unhatched blastocysts from Large White hyperprolific gilts (n=103) were identified, measured and vitrified using the Open Pulled Straw (OPS) technique to evaluate the effects of the collected blastocyst size and cryoprotectant concentrations used for vitrification, and the number of embryos transferred per recipient. Vitrified/warmed blastocyst viability was estimated in vitro, as the percentage of embryos developing after 72h, and in vivo, on pregnancy Day 30. In the in vitro study, we compared the use of three cryoprotectant concentrations (16.5, 18, or 20% DMSO+16.5, 18, or 20% EG+0.4M sucrose). Survival rates differed significantly between the control (98.3%) and the three cryoprotectant concentrations (67, 62.3, and 57%, respectively). Blastocyst size at vitrification determined the further in vitro development of embryos (26% survival for blastocysts 126-144microm versus 100% for blastocysts >199microm). For the in vivo study, blastocysts were vitrified using cryoprotectant concentrations of 16.5 or 18% DMSO+EG and transferred surgically in groups of 20 or 30 per recipient (n=40). Recipients were slaughtered on pregnancy D30. No significant differences were detected in gestation rates (50-70%) and embryo survival rates (14.7-25%), although survival was higher (P=0.0003) when 20 blastocysts were transferred compared to 30 (24.7% versus 15.5%). Our findings indicate that best results, in terms of subsequent in vivo embryo survival, were achieved after transferring 20 embryos at the blastocyst or expanded blastocyst stage, previously vitrified using cryoprotectant concentrations of 16.5 or 18%.

The effectiveness of the stereomicroscopic evaluation of embryo quality in vitrified-warmed porcine blastocysts: an ultrastructural and cell death study.

The objective of this study was to analyze the validity of the stereomicroscopic evaluation of vitrified-warmed (V-W) porcine blastocysts. Unhatched blastocysts were obtained from Large-white gilts (n=10). Blastocysts (n=156) were vitrified using the Open Pulled Straw technology. After warming, V-W blastocysts were cultured for 24h (V24). Then, their developmental progression was morphologically assessed by stereomicroscopy and classified as: V24 viable re-expanded blastocysts; V24 viable hatched blastocysts or V24 degenerated. Blastocysts which re-expanded or hatched after warming were considered viable. Some fresh blastocysts were not vitrified and were evaluated after 24h in culture (F24). By stereomicroscopic analysis all the fresh blastocysts were considered viable. Some F24, V24 re-expanded viable, V24 hatched viable and V24 degenerated blastocysts were processed for transmission electron microscopy (n=13, 19, 9 and 9, respectively) or assessed by TUNEL for cell-death evaluation (n=16, 21, 11 and 21, respectively). All V24 hatched blastocysts showed similar ultrastructure to fresh blastocysts. However, some V24 re-expanded blastocysts considered viable (6/19) revealed ultrastructural alterations. Degenerated V24 blastocysts showed ultrastructural disintegration. Hatched V24 blastocysts did not differ (p>0.05) from F24 hatched blastocysts with regard to the ratio of dead cells (2.8+/-0.5% versus 1.9+/-0.3%, respectively). However, V24 expanded blastocysts had higher (p<0.01) cell death levels (4.3+/-3.4%) than those observed in the F24 expanded blastocysts (1.1+/-0.3%). The degenerated blastocysts showed the highest cell-death index (19.4+/-6.3%). In summary, V-W blastocyst hatching during in vitro culture appears to coincide with good ultrastructure and low cell-death index, suggesting that the hatching rate assessed by stereomicroscopy is more appropriate than embryo re-expansion for an evaluation of V-W blastocyst quality.

Piglets born after non-surgical deep intrauterine transfer of vitrified blastocysts in gilts.

The aims of this study were: (1) to evaluate the effect of the number of previous estrus of recipient gilts on effectiveness of intrauterine insertion of a flexible catheter designed for non-surgical deep intrauterine catheterization during diestrus in pigs; and (2) to determine the farrowing rate and the litter size after non-surgical deep intrauterine embryo transfer (ET) of porcine blastocysts vitrified by the open pulled straw (OPS) method. In experiment 1, 27 large white hyperprolific gilts (LWh) with 2-6 previous estrus were used. Intrauterine insertions of the flexible catheter were carried out at day 5.5-6 of the estrous cycle (D0=onset of estrus). During insertions, no or only moderate reactions were observed in 88.9% of gilts and was not related (P >0.05) to the number of estrus prior to the insertion periods. The number of the estrus had a significant effect (P <0.05) on the difficulties found during the procedure. In the 100% of gilts with two estrus (N=6) it was not possible to insert the flexible catheter through the cervix. In gilts with three or more estrus, it was possible to pass the cervix and to progress along a uterine horn in 80.9% of the cases. In 86.7% of the gilts, the tip of the flexible catheter achieved the second or third quarter of the uterine horn. In experiment 2, following non-surgical deep intrauterine transfer of 20 vitrified/warmed blastocysts, 9 Meishan recipients (42.9%) farrowed an average of 5.4 +/- 0.8 piglets (range 3-9) of which 0.6 +/- 0.3 piglets (range 0-2) were born dead. In conclusion, this study shows that it is possible to obtain birth of piglets following non-surgical deep intrauterine embryo transfer (ET) of vitrified/warmed blastocysts. Non-surgical deep intrauterine ET and OPS vitrification methods are promising procedures to be used together for the introduction of new genetic material in a farm.

Flow cytometric quantification of HIV-1 Tat protein in tat-transfected Jurkat T cell lines.

The transactivator Tat protein represents a pivotal factor for the replication of human immunodeficiency virus type 1 (HIV-1). In this report, we describe a flow cytometry procedure designed to quantify the intracellular content of Tat protein in Jurkat CD4+ T lymphoblastoid cell lines, stably transfected with plasmids expressing full-length Tat protein. Various expression vectors were compared for their effectiveness to yield Tat protein in Jurkat cells, and several technical parameters were analyzed to optimize the assay. This method offers a quick and efficient approach to select stably transfected cell lines expressing different levels of specific protein.

The corneal endothelium after myopic excimer laser photorefractive keratectomy.

OBJECTIVE: To investigate whether 193-nm excimer laser photorefractive keratectomy for the correction of myopia can determine in vivo changes in the human corneal endothelium 3 and 12 months after surgery. METHODS: Contact wide-field specular biomicroscopy of the central corneal endothelium was performed on 76 eyes of 61 patients (aged 20 to 49 years; mean, 29.7 years) who underwent photorefractive keratectomy for the correction of myopia ranging between -1.75 and -13.50 diopters (mean, -6.60 D). The maximum estimated central depth of the photoablation was 113 microns. Photomicrographs of the endothelium taken before surgery were processed by means of computer-assisted morphometric analysis and compared with those obtained 3 and 12 months after surgery. RESULTS: The mean (+/- SD) cell density was 2657 +/- 298 cells/mm2 preoperatively, 2672 +/- 286 cells/mm2 after 3 months (P = .670), and 2656 +/- 289 cells/mm2 after 1 year (P = .982). The mean (+/- SD) coefficient of cell area variation was 30.27% +/- 5.99% preoperatively, 28.25% +/- 5.21% after 3 months (P = .032), and 26.35% +/- 5.29% after 1 year (P = .001). The mean (+/- SD) percentage of hexagonal cells was 63.82% +/- 9.94% preoperatively, 64.02% +/- 10.53% after 3 months (P = .806), and 67.19% +/- 9.14% after 1 year (P = .025). CONCLUSIONS: Photorefractive keratectomy caused no damage to the corneal endothelium. Postoperative improvements of the coefficient cell area variation and the percentage of hexagonal cell values may be related to corneal metabolic modifications or discontinuance of contact lens use after surgery.

Corneal endothelial anomalies in the fetal alcohol syndrome.

The fetal alcohol syndrome involves various neural crest-derived structures thus causing systemic and ocular malformations. This study investigated anomalies of the corneal endothelium, a neural crest-derived tissue, in eight children affected by fetal alcohol syndrome without known anterior segment anomalies. We performed specular biomicroscopy on the central corneal endothelium. The data were then compared with those from 80 age-matched healthy children, applying the same methods. Significant differences were found between patients with fetal alcohol syndrome and healthy subjects for mean cell density (P = .032), polymegethism (P = .000), and percentage of hexagonal cells (P = .000). We also found a close correlation between endothelial anomalies and auditory dysfunction in the patients with fetal alcohol syndrome. These alterations may be a consequence of alcohol-induced toxic effects on neural crest cells destined to form both the corneal endothelium and the organ of Corti in the same embryogenic period.

Mycobacterium chelonae keratitis after excimer laser photorefractive keratectomy.

This is the first report of a severe case of Mycobacterium chelonae keratitis; it occurred in a 26-year-old man after he had undergone excimer laser photorefractive keratectomy for the correction of severe myopia, once the epithelium was already healed. The diagnosis was made by culture results and acid-fast staining of corneal scrapings. Topical ciprofloxacin sodium, 0.3 mg/mL, plus amikacin sodium, 10 mg/mL, and oral clarithromycin sodium led to remission of the ulceration after 3 months of therapy. Subsequent topical corticosteroid therapy led to complete visual recovery during 1 year of follow-up. There may be an increased risk of severe keratitis during the first postoperative months in eyes that have already undergone photorefractive keratectomy, due to the presence of some microepithelial defects symptomatically negative and not easily detectable by slit-lamp examination.

Photorefractive keratectomy for compound myopic astigmatism with an eye cup erodible mask delivery system.

BACKGROUND: The erodible mask is a new energy delivery system for the 193-nm argon fluoride excimer laser. It consists of a polymethyl-methacrylate button, whose profile is transferred by photoablation onto the corneal surface. We present the 6- and 12-month results of this technique in the correction of compound myopic astigmatism. METHODS: We performed the mask procedure on 21 eyes of 16 subjects (mean age, 30.7 years; range, 24 to 46) to correct combined myopia and astigmatism. Attempted myopic correction ranged between -1.50 diopters (D) and -10.00 D (mean, -7.07 D). Attempted astigmatic correction ranged between -1.50 D and -4.00 D (mean, -2.46 D). RESULTS: Mean procedure error was: sphere +0.74 D (range, -3.00/+5.00), cylinder -1.41 D (range, -3.50/0.00) at 1 month after surgery; sphere +0.18 D (range, -2.50/+ 3.50), cylinder -1.56 D (range, -4.00/0.00) at 6 months; and sphere -1.30 D (range, -3.00/0.00), cylinder was -1.25 D (range, -2.00/-0.50) at 12 months (10 eyes). During follow up, haze values were never higher than 1, except for one case of haze 2 that regressed to 0 during follow up. Postoperative uncorrected visual acuity improved in all eyes where emmetropia was envisaged; none of the eyes lost spectacle-corrected visual acuity lines 6 or 12 months after surgery. CONCLUSIONS: The erodible mask proved effective and fairly predictable mainly in the correction of the spherical component of refractive error, while the correction of astigmatism revealed greater unpredictability, with a constant trend to undercorrection.

Photorefractive keratectomy for hyperopia using an erodible disc and axicon lens: 2-year results.

BACKGROUND: This paper presents the results over a 2-year follow-up of the first human trial of photorefractive keratectomy (PRK) for correction of hyperopia using an erodible disc excimer laser delivery system (Summit) coupled to an axicon lens. METHODS: We treated 25 eyes of 21 patients for a mean correction of +3.38 +/- 0.97 D (range, +1.00 to +4.00 D). The hyperopic correction was made using an erodible disc inserted on the laser optical pathway; an axicon lens was then used to create a blend transition zone. Eyes were evaluated at 1, 3, 6, and 12 months after surgery. For a smaller series of 11 eyes, we also present 24-month results. RESULTS: Mean refractive error 1 month after treatment (25 eyes) was -2.35 +/- 1.55 D (range, +1.00 to -6.50 D). Eight eyes (32%) had a spectacle-corrected visual acuity loss greater than 1 line. Twelve months after treatment, mean spherical equivalent refraction was -0.47 +/- 0.80 D (range, +1.25 to -2.25 D). Nineteen eyes showed an improvement (range, 3 to 8 lines) in uncorrected distance visual acuity and 23 showed improvement in uncorrected vision at reading distance (1 to 7 lines). CONCLUSION: This technique proved effective in reducing hyperopia, but predictability must be demonstrated in a larger treatment group. Safety was confirmed by the absence of delayed reepithelialization and the absence of spectacle-corrected visual acuity loss greater than 1 line at 1 year after surgery.

Automatic eye tracker for excimer laser photorefractive keratectomy.

Acute overlapping of successive laser pulses onto the cornea during photorefractive keratectomy (PRK) is important to avoid refractive distortions. Most excimer laser systems performing corneal ablation lack control of the patient's eye movements and they cannot track the target corneal zone. We developed an eye-tracker based on television monitoring of the pupil and on automatic electro-mechanical deflection of the laser turning mirror, and we applied it to the ExciMed UV200 ArF work station (Summit Technology, Inc., Waltham, Mass.). Basic components are a black and white CCD camera and two fast stepping motors. The circuitry for target discrimination and tracking, and the optical imaging system are designed specifically. The tracker assembly does not interfere with the laser beam path nor with the operator's observation. Tracking of the pupil has been successfully achieved on different color irides, with an accuracy better than 0.1 mm in a 6 x 6 mm2 tracking field. Response time is less than 100 ms. Recordings of eye movements during PRK are presented. Tracking ablations have been performed on moving test eye-balls with plastic corneas. The proposed automatic system appears to be a reliable and effective method for the compensation of patient eye movements appears to be a reliable and effective method during PRK procedures.

Meiotic and developmental competence of prepubertal and adult swine oocytes.

The present study was conducted to compare meiotic and cytoplasmic competence of prepubertal and adult porcine oocytes, and the effects of EGF (0 to 100 ng/mL), FSH (0 to 400 ng/mL) and prepubertal pFF (0 to 10%) on nuclear maturation. Prepubertal oocytes were less responsive to FSH and pFF than were adult oocytes in terms of stimulation of nuclear maturation. The best nuclear maturation rates for prepubertal oocytes were obtained with 10 ng/mL EGF and 400 ng/mL FSH, whereas for adult oocytes no additional effect of EGF was seen in the presence of 400 ng/mL FSH. Supplementation with pFF had no additional effect on MII yield over that obtained with EGF plus FSH. After maturation in the presence of EGF, FSH and cysteamine, fertilization rates were not different between adult and prepubertal oocytes, but polyspermy was more frequent in prepubertal oocytes (31 +/- 17% vs. 17 +/- 7% in prepubertal and adult oocytes, respectively, P < 0.05). The addition of pFF to maturation medium decreased oocyte fertilization of adult oocytes and polyspermic fertilization in prepubertal oocytes. Blastocyst yield and developmental competence were significantly reduced in prepubertal oocytes compared to adult oocytes. The mean cell numbers in blastocysts cultured for 7 days ranged from 61 to 74, and did not differ among groups. Finally, the viability of the 2- to 4-cell embryos and blastocysts produced was assessed by embryo transfer experiments. One offspring was obtained after transfer of 2- to 4-cell embryos, and one after transfer of in vitro-produced blastocysts. In conclusion, although prepubertal gilt oocytes appeared less meiotically and developmentally competent than their adult counterparts, they can be used to produce blastocysts able to develop to term.

The human corneal endothelium after myopic excimer laser photorefractive keratectomy immediate to one-month follow-up.

PURPOSE: To assess whether excimer laser photorefractive keratectomy for myopia (PRK) induced any changes in the human corneal endothelium in the immediate postoperative period and up to one month after surgery. METHODS: No-contact wide-field specular biomicroscopy of the central corneal endothelium was performed on 56 eyes of 56 patients (age 20-54 years, mean 31.3) who underwent photorefractive keratectomy for the correction of myopia ranging between 1.25 and 12.50 D (5.71 +/- 2.96 D, mean +/- SD). Images of the endothelium were obtained using the NonCon Robo endothelial biomicroscope (Konan, Hyogo, Japan), and processed to calculate the mean cell density (MCD), the coefficient of variation of cell area (CV), and the percentage of hexagonal cells (Hex%). Eyes were evaluated immediately before (preop) and after surgery (day 0), on the first two postoperative days (days 1 and 2), and after one month. RESULTS: MCD was 2645 +/- 344 cells/mm2 at preop, 2609 +/- 293 cells/mm2 on day 0, 2681 +/- 320 cells/mm2 on day 1, 2629 +/- 305 cells/mm2 on day 2, and 2709 +/- 308 cells/mm2 at one month; a significant difference was found only comparing preoperative to one month data (p = 0.009). CV was 31.6 +/- 4.7 at preop, 39 +/- 7.1 on day 0 (p = 0.000001), 37.1 +/- 7.8 on day 1 (p = 0.00005), 43.4 +/- 13.1 on day 2 (p = 0.000001), and 34.7 +/- 5.8 on one month (p = 0.0006). Hex% was 61.9 +/- 7.1 at preop, 58 +/- 7.3 on day 0 (p = 0.002), 58.5 +/- 8.5 on day 1 (p = 0.015), 57.1 +/- 8.5 on day 2 (p = 0.00004), and 59.3 +/- 7.5 at one month (p = 0.002). No correlations were found between ablation depth and endothelial changes. CONCLUSIONS: Endothelial cell density was not reduced by PRK in the immediate postoperative period. Changes in morphological indices like CV and Hex% indicate that the photoablation causes some kind of damage to the endothelial cells.

Compound myopic astigmatism correction using a mask in-the-rail excimer laser delivery system. Preliminary results.

PURPOSE: The latest development in the erodible mask technology is an excimer laser containing the mask in the laser optical pathway. This paper reports the results of the first human series of consecutive treatments performed for the correction of compound myopic astigmatism. METHODS: We have treated 83 eyes. Spherical equivalent attempted correction ranged between -1.75 and -11.75 D (mean -7.07+/- 2.45 D), astigmatic attempted correction ranged between -1.00 and -5.00 D (mean -2.42+/- 1.02 D). The sphere correction was made by diaphragm using a multi-zone software with three ablation zones: 100% of the total attempted correction for the central 5.0 mm zone, 70% for the second 6.0 mm zone, 30% for the outer 6.5 mm zone. Cylinder correction was made sequentially after myopic correction using the appropriate mask. RESULTS: One month after treatment, mean refractive error was + 1.07 +/- 1.24 D (range + 4.50/-1.75 D) for spherical equivalent, and -0.49 +/- 0.57 D (range + 0.75/-2.00 D) for astigmatism. Forty-eight eyes (57.8%) had uncorrected visual acuity of 20/40 or better. At six months, mean refractive error was +0.42 +/- 0.97 D (range + 3.75/-1.00 D) for spherical equivalent, and -0.44 +/- 0.51 D (range +0.25/-3.00 D) for astigmatism. Vector analysis showed that 57 eyes (68.7%) had 5 or less degrees rotation. Seventy-two (86.7%) and 54 eyes (65.1%) had uncorrected visual acuity equal or better than 20/40 and 20/25 respectively. One eye (1.2%) showed a best corrected visual acuity loss of more than one line, but 8 eyes (9.6%) had a gain of more than one line. One-year results on a smaller series (33 eyes) overlap with the six-month results. CONCLUSIONS: The mask in-the-rail excimer laser delivery system appears to be effective and predictable in the correction of compound myopic astigmatism. We observed no significant regression of the astigmatic correction over time. Longer follow-up on larger series is necessary to draw final conclusions.

Hyperopia correction using an erodible mask excimer laser delivery system coupled to an axicon: preliminary results.

PURPOSE: This paper presents the results of the first human trial on the correction of hyperopia using an erodible mask excimer laser delivery system coupled to an axicon. METHODS: We treated 17 eyes of 17 patients (age range 34-62 years) for the correction of +3.21 +/- 1.04 D (range +1.00 to +4.00 D). The hyperopic correction was made using an erodible mask inserted on the laser optical pathway, to produce a circular ablation measuring 6.5 mm in diameter. An axicon was then used to create a blend transition zone from 6.5 mm up to 9.4 mm in diameter. Eyes were evaluated at one, three and six months after surgery. RESULTS: Reepithelization was always observed by the fifth postoperative day, despite the large area of deepithelization (diameter 9.5 mm). Mean refractive error one month after treatment was -2.44 +/- 1.59 D (range 0.00 to -6.50 D). Five eyes (29.4%) had a best corrected visual acuity loss more than two to three lines; all eyes showed mild annular haze not involving the central part of the cornea. Six months after treatment, mean refractive error was -0.88 +/- 0.99 D (range +0.50 to -3.00 D). Compared to preoperative status, 13 eyes (76.5%) showed an improvement in uncorrected distance visual acuity (1-8 lines), and 14 eyes (82.4%) showed an improvement in uncorrected vision at reading distance (3-7 lines). Two eyes (11.7%) showed a best corrected visual acuity loss of two of three lines. CONCLUSIONS: These preliminary results indicate this approach is effective in reducing hyperopia, while its predictability has still to be proved in a larger treatment group with longer follow-up. A cautious approach to this technique is still advisable, especially for higher hyperopic corrections, in view of the large best corrected visual acuity loss seen in two eyes at six months.