Measurements of cerebral microvascular blood flow, oxygenation, and morphology in a mouse model of whole-brain irradiation-induced cognitive impairment by two-photon microscopy and optical coherence tomography: evidence for microvascular injury in the cerebral white matter.

Whole-brain irradiation (WBI, also known as whole-brain radiation therapy) is a mainstay treatment modality for patients with multiple brain metastases. It is also used as a prophylactic treatment for microscopic tumors that cannot be detected by magnetic resonance imaging. WBI induces a progressive cognitive decline in ~ 50% of the patients surviving over 6 months, significantly compromising the quality of life. There is increasing preclinical evidence that radiation-induced injury to the cerebral microvasculature and accelerated neurovascular senescence plays a central role in this side effect of WBI. To better understand this side effect, male C57BL/6 mice were first subjected to a clinically relevant protocol of fractionated WBI (5 Gy, two doses per week, for 4 weeks). Nine months post the WBI treatment, we applied two-photon microscopy and Doppler optical coherence tomography to measure capillary red-blood-cell (RBC) flux, capillary morphology, and microvascular oxygen partial pressure (PO2) in the cerebral somatosensory cortex in the awake, head-restrained, WPI-treated mice and their age-matched controls, through a cover-glass-sealed chronic cranial window. Thanks to the extended penetration depth with the fluorophore - Alexa680, measurements of capillary blood flow properties (e.g., RBC flux, speed, and linear density) in the cerebral subcortical white matter were enabled. We found that the WBI-treated mice exhibited a significantly decreased capillary RBC flux in the white matter. WBI also caused a significant reduction in capillary diameter, as well as a large (although insignificant) reduction in segment density at the deeper cortical layers (e.g., 600-700 µm), while the other morphological properties (e.g., segment length and tortuosity) were not obviously affected. In addition, we found that PO2 measured in the arterioles and venules, as well as the calculated oxygen saturation and oxygen extraction fraction, were not obviously affected by WBI. Lastly, WBI was associated with a significant increase in the erythrocyte-associated transients of PO2, while the changes of other cerebral capillary PO2 properties (e.g., capillary mean-PO2, RBC-PO2, and InterRBC-PO2) were not significant. Collectively, our findings support the notion that WBI results in persistent cerebral white matter microvascular impairment, which likely contributes to the WBI-induced brain injury and cognitive decline. Further studies are warranted to assess the WBI-induced changes in brain tissue oxygenation and malfunction of the white matter microvasculature as well.

Prevalence of class I-III BRAF mutations among 114,662 cancer patients in a large genomic database.

IMPACT STATEMENT: These data represent the largest aggregation of BRAF mutations within a single clinical database to our knowledge. The relative proportions of both BRAF V600 mutations and non-V600 mutations are informative in all cancers and by malignancy, and can serve as a definitive gold-standard for BRAF mutation cancer incidence by malignancy. The rate of BRAF mutation in human cancer in a real-world large database is lower than previously reported likely representing testing more broadly across tumor types. The relative percentages of Class II and Class III BRAF mutations are higher than previously reported, representing almost 35% of BRAF mutations in cancer. These findings provide support for the development of effective treatments for non-V600 BRAF mutations in cancer.

Immunotherapeutic approaches for small-cell lung cancer.

Immune-checkpoint inhibitors (ICIs) are approved in the first-line and third-line settings for patients with extensive-stage or relapsed small-cell lung cancer (SCLC), respectively. In the first-line setting, the addition of the anti-programmed cell death 1 ligand 1 (PD-L1) antibody atezolizumab to chemotherapy improves overall survival (OS). In patients with relapsed disease, data from nonrandomized trials have revealed promising responses, although a significant improvement in OS over that obtained with conventional chemotherapy was not achieved in a randomized trial in this setting. Substantial research interest exists in identifying predictive biomarkers that could guide the use of ICIs in patients with SCLC. PD-L1 expression is typically low or absent in SCLC, which has precluded its use as a predictive biomarker. Tumour mutational burden might have some predictive value, although blood-based measures of tumour mutational burden did not have predictive value in patients receiving atezolizumab plus chemotherapy in the first-line setting. After three decades, ICIs have finally enabled an improvement in OS for patients with SCLC; however, a substantial amount of research remains to be done, including identifying the optimal therapeutic strategy and predictive biomarkers. In this Review, we describe the available data on clinical efficacy, the emerging evidence regarding biomarkers and ongoing clinical trials using ICIs and other immunotherapies in patients with SCLC.

Serum Neurofilament Light, Glial Fibrillary Acidic Protein and Tau Are Possible Serum Biomarkers for Activity of Brain Metastases and Gliomas.

BACKGROUND: Primary central nervous system (CNS) tumors and brain metastases (BMs) are major causes of morbidity and mortality, accompanied by low survival rates. Efforts to early discovery of CNS malignancies are critical. However, to date, there are no biomarkers approved for detection of cancer activity in the brain. Blood levels of neurofilament light (NfL) and tau, as well as glial fibrillary acidic protein (GFAp), show promise as biomarkers for brain injury in previous studies. Therefore, we performed a cross-sectional study to investigate correlations of those biomarkers with CNS activity of gliomas and BMs. METHODS: Serum samples of 36 participants of a single centered institution were tested for NfL, GFAp and tau with Simoa immunoassay, and correlated with clinical and radiological data. RESULTS: NfL and GFAp levels were significantly associated with the state of intracranial disease (analysis of variance (ANOVA), PsNfL = 0.03; ANOVA, PGFAp = 0.03). Although statistically significant (P = 0.04), differences in concentrations were not clinically meaningful for tau levels. Serum NfL (sNfL) and GFAp concentrations were higher in the group of patients with CNS tumors with disease in progression versus CNS with stable disease (P = 0.03 and P = 0.01, respectively). In addition, sNfL were higher in patients with metastatic solid tumors with known BMs than in those with metastatic tumors with no BM (P = 0.0004). CONCLUSION: sNfL and GFAp both apparently vary closely with presence and activity of gliomas and BMs. Further studies in larger populations are needed to expand these findings.

Embryonic overexpression of receptors for advanced glycation end-products by alveolar epithelium induces an imbalance between proliferation and apoptosis.

Receptors for advanced glycation end-products (RAGEs) are multiligand cell surface receptors highly expressed in the lung that contribute to alveolar epithelial cell differentiation during embryogenesis and the modulation of pulmonary inflammation during disease. When RAGEs are overexpressed throughout embryogenesis, severe lung hypoplasia ensues, culminating in perinatal lethality. However, the possible mechanisms that lead to the disappearance of pulmonary tissue remain unclear. A time course of lung organogenesis, commencing on Embryonic Day (E) 12.5, demonstrated that increased RAGE expression primarily alters lung morphogenesis beginning on E16.5. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) immunohistochemistry and immunoblotting for active caspase-3 confirmed a shift toward apoptosis in lungs from RAGE-overexpressing mice, compared with wild-type control mice. This observation supports previous work where electron microscopy identified the cellular blebbing of alveolar epithelium in embryonic RAGE-overexpressing mice. Assaying for NF-κB also revealed elevated nuclear translocation in lungs from transgenic mice compared with control mice. An RT-PCR assessment of genes regulated by NF-κB demonstrated the elevated expression of Fas ligand, suggesting increased activity of the Fas-mediated signal transduction pathway in which ligand-receptor interactions trigger cell death. These data provide evidence that the expression of RAGEs must be tightly regulated during homeostatic organogenesis. Further elucidations of the RAGE signaling potentially involved in cell-cycle abnormalities may provide insights into the progression of RAGE-mediated lung diseases.

Cytochrome P450 1B1 contributes to renal dysfunction and damage caused by angiotensin II in mice.

Cytochrome P450 1B1 contributes to the development of angiotensin II-induced hypertension and associated cardiovascular pathophysiology. In view of the critical role of angiotensin II in the kidney, as well as in salt and water homeostasis, and blood pressure regulation, we determined the contribution of cytochrome P450 1B1 to renal dysfunction and injury associated with angiotensin II-induced hypertension in male Cyp1b1(+/+) and Cyp1b1(-/-) mice. Angiotensin II infusion (700 ng/kg per minute) given by miniosmotic pumps for 13 and 28 days increased systolic blood pressure in Cyp1b1(+/+) mice; this increase was significantly reduced in Cyp1b1(-/-) mice. Angiotensin II increased renal Cyp1b1 activity, vascular resistance, and reactivity to vasoconstrictor agents and caused endothelial dysfunction in Cyp1b1(+/+) but not Cyp1b1(-/-) mice. Angiotensin II increased water consumption and urine output, decreased urine osmolality, increased urinary Na(+) and K(+) excretion, and caused proteinuria and albuminuria in Cyp1b1(+/+) mice that was diminished in Cyp1b1(-/-) mice. Infusion of angiotensin II for 28 but not 13 days caused renal fibrosis, tubular damage, and inflammation in Cyp1b1(+/+) mice, which was minimized in Cyp1b1(-/-) mice. Angiotensin II increased levels of 12- and 20-hydroxyeicosatetraenoic acids; reactive oxygen species; and activity of NADPH oxidase, extracellular signal-regulated kinase 1/2, p38 mitogen-activated protein kinase, and c-Src in the kidneys of Cyp1b1(+/+) but not Cyp1b1(-/-) mice. These data suggest that increased thirst, renal dysfunction, and injury and inflammation associated with angiotensin II-induced hypertension in mice depend on cytochrome P450 1B1 activity, thus indicating that cytochrome P450 1B1 could serve as a novel target for treating renal disease and hypertension.

Evaluating the global CpG methylation status of native DNA utilizing a bipartite split-luciferase sensor.

Epigenetic modifications play an essential role in the regulation of gene expression and ultimately cell fate. Methylation of cytosine at CpG dinucleotides (mCpG) is an important epigenetic mark that has been correlated with cancer when present at promoter sites of tumor suppressor genes. To develop a rapid methodology for the direct assessment of global levels of DNA methylation, we first interrogated the methyl-CpG binding domains (MBDs), the Kaiso family of Cys(2)-His(2) zinc fingers, and an SET- and RING-associated domain using a split-luciferase reassembly methodology. We identified MBD1 as the most selective domain for the discrimination between mCpG and CpG sites with over 90-fold selectivity. Utilizing a bipartite strategy, we constructed a purely methylation-dependent bipartite sensor for the direct detection of global levels of DNA methylation by attaching MBD1 domains to each of the split-luciferase halves. This new sensor was validated for the direct determination of genomic DNA methylation levels in in vitro studies without any intervening chemical or enzymatic processing of DNA. Finally, we demonstrated that this bipartite sensor can be utilized for monitoring dose-dependent changes in global levels of methylation in DNA from HeLa cells challenged with 5-aza-2'-deoxycytidine, a DNA methyltransferase inhibitor.

Immunohistochemical detection and regulation of α5 nicotinic acetylcholine receptor (nAChR) subunits by FoxA2 during mouse lung organogenesis.

BACKGROUND: α5 nicotinic acetylcholine receptor (nAChR) subunits structurally stabilize functional nAChRs in many non-neuronal tissue types. The expression of α5 nAChR subunits and cell-specific markers were assessed during lung morphogenesis by co-localizing immunohistochemistry from embryonic day (E) 13.5 to post natal day (PN) 20. Transcriptional control of α5 nAChR expression by FoxA2 and GATA-6 was determined by reporter gene assays. RESULTS: Steady expression of α5 nAChR subunits was observed in distal lung epithelial cells during development while proximal lung expression significantly alternates between abundant prenatal expression, absence at PN4 and PN10, and a return to intense expression at PN20. α5 expression was most abundant on luminal edges of alveolar type (AT) I and ATII cells, non-ciliated Clara cells, and ciliated cells in the proximal lung at various periods of lung formation. Expression of α5 nAChR subunits correlated with cell differentiation and reporter gene assays suggest expression of α5 is regulated in part by FoxA2, with possible cooperation by GATA-6. CONCLUSIONS: Our data reveal a highly regulated temporal-spatial pattern of α5 nAChR subunit expression during important periods of lung morphogenesis. Due to specific regulation by FoxA2 and distinct identification of α5 in alveolar epithelium and Clara cells, future studies may identify possible mechanisms of cell differentiation and lung homeostasis mediated at least in part by α5-containing nAChRs.

Profiling small molecule inhibitors against helix-receptor interactions: the Bcl-2 family inhibitor BH3I-1 potently inhibits p53/hDM2.

We validate a practical methodology for the rapid profiling of small molecule inhibitors of protein-protein interactions. We find that a well known BH3 family inhibitor can potently inhibit the p53/hDM2 interaction.

Why preoperative acuity predicts postoperative acuity in wavefront-guided LASIK.

PURPOSE: To critically evaluate the following clinical wisdom regarding custom (wavefront-guided) laser in situ keratomileusis (LASIK) that subjects with better-than-average best-corrected visual acuity (BCVA) before surgery have a greater risk of losing BCVA postoperatively than do subjects with worse-than-average BCVA before surgery. METHODS: High contrast BCVA was measured once before and 3 months after custom LASIK in one eye of 79 subjects. Preoperative spherical equivalent refractive error ranged between -1.00 and -10.38 D. The sample was divided into one of two subsamples: eyes that had better-than-average preoperative BCVA (<-0.11 logMAR) and eyes that had average or worse-than-average preoperative BCVA (≥-0.11 logMAR). Controls were implemented for retinal magnification and for the statistical phenomenon of regression to the mean of the preoperative acuity measurement. RESULTS: On average, for the entire sample, moving the correction from the spectacle plane to the corneal plane increased letter acuity 4.7% (1 letter, 0.02 logMAR). For each subsample, the percentage regression to the mean was 57.24%. After correcting for magnification effects and regression to the mean, eyes with better-than-average preoperative acuity had a small but significant gain in acuity (∼1 letter, p = 0.040) that was nearly identical to the gain for eyes with worse-than-average preoperative acuity (∼1.5 letters, p = 0.002). CONCLUSIONS: Custom LASIK produced a statistically significant gain in visual acuity after correction for magnification effects. Dividing the sample into two subsamples based on preoperative acuity confirmed the common clinical observation that eyes with better-than-average acuity tend to remain the same or lose acuity, whereas eyes with worse-than-average acuity tend to gain acuity. However, when only one acuity measurement is taken at a single time point and the sample is subsampled nonrandomly, this clinical observation is due to a statistical artifact (regression to the mean) and is not attributable to the surgery.

A general approach for receptor and antibody-targeted detection of native proteins utilizing split-luciferase reassembly.

The direct detection of native proteins in heterogeneous solutions remains a challenging problem. Standard methodologies rely on a separation step to circumvent nonspecific signal generation. We hypothesized that a simple and general method for the detection of native proteins in solution could be achieved through ternary complexation, where the conditional signal generation afforded by split-protein reporters could be married to the specificity afforded by either native receptors or specific antibodies. Toward this goal, we describe a solution phase split-luciferase assay for native protein detection, where we fused fragmented halves of firefly luciferase to separate receptor fragments or single-chain antibodies, allowing for conditional luciferase complementation in the presence of several biologically significant protein targets. To demonstrate the utility of this strategy, we have developed and validated assay platforms for the vascular endothelial growth factor, the gp120 coat protein from HIV-1, and the human epidermal growth factor receptor 2 (HER2), a marker for breast cancer. The specificities of the recognition elements, CD4 and the 17b single-chain antibody, employed in the gp120 sensor allowed us to parse gp120s from different clades. Our rationally designed HER2 sensing platform was capable of discriminating between HER2 expression levels in several tumor cell lines. In addition, luminescence from reassembled luciferase was linear across a panel of cell lines with increasing HER2 expression. We envision that the proof of principle studies presented herein may allow for the potential detection of a broad range of biological analytes utilizing ternary split-protein systems.

Toward a general approach for RNA-templated hierarchical assembly of split-proteins.

The ability to conditionally turn on a signal or induce a function in the presence of a user-defined RNA target has potential applications in medicine and synthetic biology. Although sequence-specific pumilio repeat proteins can target a limited set of ssRNA sequences, there are no general methods for targeting ssRNA with designed proteins. As a first step toward RNA recognition, we utilized the RNA binding domain of argonaute, implicated in RNA interference, for specifically targeting generic 2-nucleotide, 3' overhangs of any dsRNA. We tested the reassembly of a split-luciferase enzyme guided by argonaute-mediated recognition of newly generated nucleotide overhangs when ssRNA is targeted by a designed complementary guide sequence. This approach was successful when argonaute was utilized in conjunction with a pumilio repeat and expanded the scope of potential ssRNA targets. However, targeting any desired ssRNA remained elusive as two argonaute domains provided minimal reassembled split-luciferase. We next designed and tested a second hierarchical assembly, wherein ssDNA guides are appended to DNA hairpins that serve as a scaffold for high affinity zinc fingers attached to split-luciferase. In the presence of a ssRNA target containing adjacent sequences complementary to the guides, the hairpins are brought into proximity, allowing for zinc finger binding and concomitant reassembly of the fragmented luciferase. The scope of this new approach was validated by specifically targeting RNA encoding VEGF, hDM2, and HER2. These approaches provide potentially general design paradigms for the conditional reassembly of fragmented proteins in the presence of any desired ssRNA target.

In vivo imaging of microscopic structures in the rat retina.

PURPOSE: The ability to resolve single retinal cells in rodents in vivo has applications in rodent models of the visual system and retinal disease. The authors have characterized the performance of a fluorescence adaptive optics scanning laser ophthalmoscope (fAOSLO) that provides cellular and subcellular imaging of rat retina in vivo. METHODS: Enhanced green fluorescent protein (eGFP) was expressed in retinal ganglion cells of normal Sprague-Dawley rats via intravitreal injections of adeno-associated viral vectors. Simultaneous reflectance and fluorescence retinal images were acquired using the fAOSLO. fAOSLO resolution was characterized by comparing in vivo images with subsequent imaging of retinal sections from the same eyes using confocal microscopy. RESULTS: Retinal capillaries and eGFP-labeled ganglion cell bodies, dendrites, and axons were clearly resolved in vivo with adaptive optics. Adaptive optics correction reduced the total root mean square wavefront error, on average, from 0.30 microm to 0.05 microm (measured at 904 nm, 1.7-mm pupil). The full width at half maximum (FWHM) of the average in vivo line-spread function (LSF) was approximately 1.84 microm, approximately 82% greater than the FWHM of the diffraction-limited LSF. CONCLUSIONS: With perfect aberration compensation, the in vivo resolution in the rat eye could be approximately 2x greater than that in the human eye because of its large numerical aperture (approximately 0.43). Although the fAOSLO corrects a substantial fraction of the rat eye's aberrations, direct measurements of retinal image quality reveal some blur beyond that expected from diffraction. Nonetheless, subcellular features can be resolved, offering promise for using adaptive optics to investigate the rodent eye in vivo with high resolution.

A general and rapid cell-free approach for the interrogation of protein-protein, protein-DNA, and protein-RNA interactions and their antagonists utilizing split-protein reporters.

Split-protein reporters have emerged as a powerful methodology for imaging biomolecular interactions which are of much interest as targets for chemical intervention. Herein we describe a systematic evaluation of split-proteins, specifically the green fluorescent protein, beta-lactamase, and several luciferases, for their ability to function as reporters in completely cell-free systems to allow for the extremely rapid and sensitive determination of a wide range of biomolecular interactions without the requirement for laborious transfection, cell culture, or protein purification (12-48 h). We demonstrate that the cell-free split-luciferase system in particular is amenable for directly interrogating protein-protein, protein-DNA, and protein-RNA interactions in homogeneous assays with very high sensitivity (22-1800 fold) starting from the corresponding mRNA or DNA. Importantly, we show that the cell-free system allows for the rapid (2 h) identification of target-site specificity for protein-nucleic acid interactions and in evaluating antagonists of protein-protein and protein-peptide complexes circumventing protein purification bottlenecks. Moreover, we show that the cell-free split-protein system is adaptable for analysis of both protein-protein and protein-nucleic acid interactions in artificial cell systems comprising water-in-oil emulsions. Thus, this study provides a general and enabling methodology for the rapid interrogation of a wide variety of biomolecular interactions and their antagonists without the limitations imposed by current in vitro and in vivo approaches.

Split beta-lactamase sensor for the sequence-specific detection of DNA methylation.

The methylation pattern of genes at CpG dinucleotide sites is an emerging area in epigenetics. Furthermore, the hypermethylation profiles of tumor suppressor genes are linked to specific tumor types. Thus, new molecular approaches for the rapid determination of the methylation status of these genes could provide a powerful method for early cancer diagnosis as well as insight into mechanisms of epigenetic regulation of genetic information. Toward this end, we have recently reported the first design of a split-protein sensor for the site-specific detection of DNA methylation. In this approach a split green fluorescent protein reporter provided a sequence-specific readout of CpG methylation. In the present work, we describe a sensitive second-generation methylation detection system that utilizes the split enzymatic reporter, TEM-1 beta-lactamase, attached to specific DNA binding elements. This system, termed mCpG-SEER-beta-Lac, shows a greater than 40-fold specificity for methylated versus nonmethylated CpG target sites. Importantly, the resulting signal enhancement afforded by the catalytic activity of split-beta-lactamase allowed for the sensitive detection of 2.5 fmol of methylated target dsDNA in 5 min. Thus, this new sensor geometry represents a 250-fold enhancement in assay time and a 2000-fold enhancement in sensitivity over our first-generation system for the detection of specific sites of DNA methylation.

Wavefront aberrations in eyes with Acrysof monofocal intraocular lenses.

PURPOSE: To characterize and measure the ocular aberrations in eyes implanted with monofocal intraocular lenses (IOLs) and to study any correlation between postoperative aberrations and surgical factors. METHODS: A Tscherning aberroscope was used to measure the wavefront aberrations of 62 eyes that had undergone phacoemulsification with the implantation of foldable monofocal Acrysof MA60BM IOLs (Alcon Laboratories Inc, Ft Worth, Tex). The Zernike coefficients, measured with a pupil diameter of 6 mm, were compared with those of a normal dataset of 82 eyes of healthy young myopes. RESULTS: Spherical aberration (Z(0)4) was the most predominant higher order aberration, with a mean value of 0.37 +/- 0.16 microm. A statistically significant linear relationship was noted between the magnitude of postoperative spherical aberration and the dioptric power of the IOL. The mean spherical aberration was 33 times more in the pseudophakic group than in normal young myopic eyes. The other major higher order aberrations were trefoil (Z(-3)3) with a mean of -0.13 +/- 0.22 microm and vertical coma (Z(-1)3) with a mean value of -0.11 +/- 0.23 microm. On average, the root-mean-square of higher order aberrations in pseudophakic eyes was 2.1 times that in a normal population of young myopic eyes. CONCLUSIONS: Eyes that undergo cataract surgery with monofocal IOL implantation suffer from significant higher order aberrations. The optical design of the IOL is most likely responsible for the increase in spherical aberration, the magnitude of which is a function of the dioptric power of the IOL.

Aberrations induced in wavefront-guided laser refractive surgery due to shifts between natural and dilated pupil center locations.

PURPOSE: To determine the aberrations induced in wavefront-guided laser refractive surgery due to shifts in pupil center location from when aberrations are measured preoperatively (over a dilated pupil) to when they are corrected surgically (over a natural pupil). SETTING: Center for Visual Science and Department of Ophthalmology, University of Rochester, Rochester, New York, USA. METHODS: Shifts in pupil center were measured between dilated phenylephrine hydrochloride (Neo-Synephrine [2.5%]) and nonpharmacological mesopic conditions in 65 myopic eyes treated with wavefront-guided laser in situ keratomileusis (Technolas 217z, Bausch & Lomb). Each patient's preoperative and 6-month postoperative wave aberrations were measured over the dilated pupil. Aberrations theoretically induced by decentration of a wavefront-guided ablation were calculated and compared with those measured 6 months postoperatively (6.0 mm pupil). RESULTS: The mean magnitude of pupil center shift was 0.29 mm +/- 0.141 (SD) and usually occurred in the inferonasal direction as the pupil dilated. Depending on the magnitude of shift, the fraction of the higher-order postoperative root-mean-square wavefront error that could be due theoretically to pupil center decentrations was highly variable (mean 0.26 +/- 0.20 mm). There was little correlation between the calculated and 6-month postoperative wavefronts, most likely because pupil center decentrations are only 1 of several potential sources of postoperative aberrations. CONCLUSIONS: Measuring aberrations over a Neo-Synephrine-dilated pupil and treating them over an undilated pupil typically resulted in a shift of the wavefront-guided ablation in the superotemporal direction and an induction of higher-order aberrations. Methods referencing the aberration measurement and treatment with respect to a fixed feature on the eye will reduce the potential for inducing aberrations due to shifts in pupil center.

Higher-order aberrations in eyes with irregular corneas after laser refractive surgery.

PURPOSE: To investigate the distribution of the eye's higher-order aberrations in postoperative laser refractive surgery patients with visual complaints and highly irregular corneal shapes. DESIGN: Retrospective case-control study. PARTICIPANTS: Thirty-three symptomatic postoperative LASIK and/or photorefractive keratectomy eyes with subjective visual complaints not corrected by spectacles more than 6 months after surgery are compared with 46 normal preoperative and 46 asymptomatic successful postoperative conventional LASIK eyes. METHODS: Postoperative wave aberrations were measured for each patient using a Shack-Hartmann wavefront sensor (Zywave, Bausch & Lomb, Rochester, NY) over a 6-mm pupil. These measurements were averaged across patients with similar corneal topographic diagnoses (central islands, decentered ablations, a new group termed baby bowties, and irregularly irregular corneas). MAIN OUTCOME MEASURES: Higher-order aberrations and corneal topography. RESULTS: The average (+/-1 standard deviation) higher-order root-mean-square (rms) wavefront error values (third, fourth, and fifth orders) for the symptomatic patients was 1.31+/-0.58 microm. This was an average of 3.46 times greater than the average magnitude of normal preoperative eyes (mean rms, 0.38+/-0.14 microm), and an average of 2.3 times greater than the average magnitude of asymptomatic successful postoperative conventional LASIK eyes (mean rms, 0.58+/-0.21 microm) over a 6-mm pupil. Higher-order rms wavefront error increased with pupil size, roughly doubling for every millimeter of increasing pupil diameter. On average, eyes with central islands (n = 6) had the most vertical coma (Z3(-1); mean, -1.35+/-0.43 microm). Eyes with central islands and decentered ablations (n = 2) also had elevated amounts of spherical aberration (Z4(0); means of 0.83+/-0.11 microm and 0.69+/-0.29 microm, respectively) compared with successful postoperative LASIK eyes (mean of 0.42+/-0.20 microm). Eyes with a topographic central baby bowtie demonstrated the most secondary astigmatism (Z4(2) and Z4(-2); mean rms, 0.56+/-0.17 microm; n = 3), despite the lowest average higher-order rms (mean, 0.84+/-0.05 microm) among symptomatic topographic subgroups. Eyes with irregularly irregular corneas had a mean higher-order rms of 1.10+/-0.39 mum. CONCLUSIONS: Symptomatic postoperative laser refractive surgery patients with irregular corneas have higher-order aberrations that are 2.3 to 3.5 times greater than asymptomatic postoperative LASIK and normal preoperative eyes, respectively. The higher-order aberrations seem to correlate with corneal topography.