Feasibility of Nonanonymous Burnout Surveys in a Large Academic Department.

BACKGROUND AND OBJECTIVES: Burnout is prevalent among clinicians and entails negative personal, professional, and organizational consequences. Assessments of burnout are typically anonymous to facilitate psychological safety. This limits the capacity of leadership to help struggling providers and reduces the level of demographic detail. Nonanonymous, confidential assessments may facilitate outreach to individuals or targeted interventions for at-risk populations. METHODS: We administered the Maslach Burnout Inventory to physician faculty and advanced practice providers in an academic department of family medicine. We identified a wellness officer within the department who served as an honest broker to keep nonanonymous survey responses confidential. Respondents had the option of taking the survey anonymously or confidentially. Anonymous respondents were allowed to withhold demographic information to ensure anonymity. RESULTS: Sixty-seven of 109 providers responded (61% response rate), with 46 (69%) doing so confidentially. Burnout rates were similar between groups: 48% among confidential respondents, and 43% among anonymous respondents (P=.71). Subscales of the MBI also showed no significant differences. Because a large proportion of anonymous respondents withheld demographic data, no demographic trends could be identified among them. Younger confidential respondents were more likely to exhibit depersonalization (P=.01). CONCLUSIONS: Most participants chose to respond confidentially. There was no significant difference in the level of burnout between confidential and anonymous respondents. Our findings refute the conventional wisdom that clinicians require anonymity to respond to burnout surveys. This finding has the potential to open a new line of inquiry regarding burnout, its drivers and potential solutions.

Impact of Social Support on Colorectal Cancer Screening among Adult Hispanics/Latinos: A Randomized Community-based Study in Central Pennsylvania.

In the United States, the five-year survival rate of colorectal cancer for Latinos is lower than it is for White, non-Latinos. Differences in survival are due, in part, to Latinos being diagnosed at a later stage. An ethnic gap in the use of colorectal cancer screening contributes to the difference in survival. We developed, implemented, and evaluated a targeted colorectal cancer screening intervention to increase colorectal cancer screening uptake by sex, ethnicity, and geography. We measured actual colorectal cancer screening uptake in both arms as a method to determine completion rates. We used a randomized, community-based, participatory design to test the impact of social support (intervention) on completion of a provider-recommended, take-home fecal immunochemical test (FIT) kit screening test among average-risk, urban, and rural Pennsylvania Latino adults age 50 and older not currently adherent to national colorectal cancer screening guidelines (n = 264). Participants in each arm attended a community-based educational program offered at eight sites. Among the 264 participants, 154 (58%) returned a completed usable FIT kit screening test. A higher return rate was observed among participants in the social support arm (66.0%) compared with the control (47.2%). Participants in the social support arm were statistically significant 2.67 times as likely to return a completed FIT kit. Of these, 27 (17.5%) had a positive FIT kit screening test result. The results of this study suggest that social support is an effective method to increase colorectal cancer screening rates among Latinos. Future studies should examine dissemination and implementation of community-based strategies among Latinos that include social support.

The art of delivering evidence-based dual antiplatelet therapy.

This review, which details 2 DAPT risk scoring systems and includes a treatment guide, can help ensure that you deliver the right treatment to the right patients.

Intrapartum antibiotics and childhood atopic dermatitis.

INTRODUCTION: Atopic dermatitis (AD) in children significantly impacts families because of medical costs, "lost" hours, and secondary characteristics such as asthma and ancillary infections. We investigate whether children delivered vaginally to women receiving intrapartum antibiotics have a greater risk of AD when younger than the age of 2 years than their counterparts. METHODS: We conducted a retrospective analysis of women who delivered child(ren) vaginally between 1996 and 2008. Women were identified as those who received intrapartum antibiotics and those who did not. Pediatric records were used to determine the incidence of AD. RESULTS: We collected data for 492 mother-child pairs. Intrapartum antibiotics were administered during 128 births; 28.9% of those children were diagnosed with AD by age 2 years (relative risk [RR], 1.03; 95% confidence interval [CI], 0.75-1.41). Factors with the greatest risk of diagnosis of AD by 2 years of age were intrapartum antibiotic exposure for >24 hours (RR, 1.99; 95% CI, 1.13-3.49), first born (RR, 1.78; 95% CI, 1.33-2.38), and higher maternal education (RR, 1.43; 95% CI, 0.99-2.06). No statistical differences in the prevalence of AD related to parental eczema, maternal group B Streptococcus status, or gestational age existed. CONCLUSIONS: Exposure to antibiotics for <24 hours during a vaginal delivery does not increase the risk of AD. Studies are needed to understand whether exposure for >24 hours during the intrapartum period increases the risk of AD.

Patient experience and use of probiotics in community-based health care settings.

OBJECTIVE: To investigate patient experience with probiotics and factors that influence probiotic use among adult patients. METHOD: Patients were invited to complete a questionnaire that assessed their experiences and opinions regarding probiotics. Questionnaires were distributed to patients seeking primary health care services at a family and community medicine practice site and a community pharmacy. Patients were invited to complete the questionnaire while awaiting the physician or waiting for prescriptions to be filled. RESULTS: Overall, 162 surveys were completed and returned (66% response rate) from patients aged 18 to 89 years of age (mean 49.5 years). Most patients (n=107; 65%) were familiar with the term "probiotic", and 49 patients (29.9%) had personally used the supplements in the past. Of those who had used probiotics, the majority (57%) had used the supplements to maintain "good gastrointestinal health" and most (59%) felt that the supplements had been beneficial. However, most (59%) had not informed their health care provider about their use of the supplements. CONCLUSION: Use of probiotic supplements is common among consumers, but may not be reported to health care providers.

A strength training program for primary care patients, central Pennsylvania, 2012.

INTRODUCTION: Primary care providers can recommend strength training programs to use "Exercise as Medicine," yet few studies have examined the interest of primary care patients in these programs. METHODS: We conducted a cross-sectional survey of primary care patients in central Pennsylvania. Interest in participating in free group-based strength training and weight control programs was assessed, in addition to patient demographics, medical history, and quality of life. RESULTS: Among 414 patients, most (61.0%) were aged 54 or older, and 64.0% were female. More patients were interested in a strength training program (55.3%) than in a weight control program (45.4%). Nearly three-quarters (72.8%) of those reporting 10 or more days of poor physical health were interested in a strength training program compared with 49.5% of those reporting no days of poor physical health. After adjusting for potential confounders, those reporting poorer physical health had 2.7 greater odds (95% confidence interval, 1.4-5.1) of being interested in a strength training program compared with those reporting better physical health. Patients with hypertension, diabetes, or high cholesterol were not more interested in a strength training program than those without these conditions. CONCLUSION: Primary care practices may consider offering or referring patients to community-based strength training programs. This study observed high levels of interest in these widely available programs. Practices may also consider screening and referring those with poorer physical health, as they may be the most interested and have the most to gain from participating.

Preliminary findings on the use of osteopathic manipulative treatment: outcomes during the formation of the practice-based research network, DO-Touch.NET.

CONTEXT: Few studies have assessed the use of osteopathic manipulative treatment (OMT) and subsequent patient-reported outcomes. OBJECTIVE: To assess the current use of OMT and associated patient-reported outcomes. DESIGN: A retrospective medical record review and a prospective observational study. SETTING: Two university-based sites and their clinics associated with the practice-based research network DO-Touch.NET. PARTICIPANTS: Patients aged 18 years or older who received OMT. MAIN OUTCOME MEASURES: Medical records from 2007 were retrospectively reviewed to identify conditions being managed with OMT. From 2008 to 2010, patients were recruited before seeing their physicians. Questionnaires were distributed to patients and physicians, and information including demographics, chief complaints, symptom severity, current and past treatments, interference of symptoms with quality of life, physical examination findings, diagnoses, OMT performed, and immediate patient response to OMT was collected. A subset of patients provided data on symptom severity and frequency and other treatments daily for the 7 days after OMT. On day 7, symptom interference with quality of life was reassessed. RESULTS: Retrospective data were collected from 2569 office visits, and prospective data were collected from 299 office visits (patient age range, 18-93 years). In the medical record review, 17 of the top 25 diagnoses (68%) were related to musculoskeletal conditions. In the prospective study, 18 of the top 24 medical diagnoses (75%) were related to musculoskeletal conditions. Immediately after OMT, patients at 271 of 296 office visits (92%) felt better or much better; those at 5 (<2%) felt worse. After 7 days, patients at 126 of 175 office visits (72%) felt better or much better, and those at 10 (6%) felt worse. Average and worst symptom severity decreased until post-OMT days 4 and 5, respectively, when severity leveled off. There was decreased interference of symptoms with quality of life from before OMT to 7 days after OMT in usual/general activities, sleep, mood, and relationships (all P≤.05). CONCLUSION: These preliminary results suggest that for adults, OMT is predominantly used for managing musculoskeletal pain conditions and is effective for short-term symptom relief. Continued surveillance of DO-Touch.NET member practice outcomes may help identify priorities for osteopathic research and define evidence-based standards for OMT practice and training.

Effects of a web-based patient activation intervention to overcome clinical inertia on blood pressure control: cluster randomized controlled trial.

BACKGROUND: Only approximately half of patients with hypertension have their blood pressure controlled, due in large part to the tendency of primary care providers (PCPs) not to intensify treatment when blood pressure values are elevated. OBJECTIVE: This study tested the effect of an intervention designed to help patients ask questions at the point of care to encourage PCPs to appropriately intensify blood pressure treatment. METHODS: PCPs and their patients with hypertension (N=500) were recruited by letter and randomized into 2 study groups: (1) intervention condition in which patients used a fully automated website each month to receive tailored messages suggesting questions to ask their PCP to improve blood pressure control, and (2) control condition in which a similar tool suggested questions to ask about preventive services (eg, cancer screening). The Web-based tool was designed to be used during each of the 12 study months and before scheduled visits with PCPs. The primary outcome was the percentage of patients in both conditions with controlled blood pressure. RESULTS: Of 500 enrolled patients (intervention condition: n=282; control condition: n=218), 418 (83.6%) completed the 12-month follow-up visit. At baseline, 289 (61.5%) of participants had controlled blood pressure. Most (411/500, 82.2%) participants used the intervention during at least 6 of 12 months and 222 (62.5%) reported asking questions directly from the Web-based tool. There were no group differences in asking about medication intensification and there were no differences in blood pressure control after 12 months between the intervention condition (201/282, 71.3%) and control condition (143/218, 65.6%; P=.27) groups. More intervention condition participants discussed having a creatinine test (92, 52.6% vs 49, 35.5%; P=.02) and urine protein test (81, 44.8% vs 21, 14.6%; P<.001), but no group differences were observed in the rate of testing. The control condition participants reported more frequent discussions about tetanus and pneumonia vaccines and reported more tetanus (30, 13.8% vs 15, 5.3%; P=.02) and pneumonia (25, 11.5% vs 16, 5.7%; P=.02) vaccinations after 12 months. CONCLUSIONS: The use of an interactive website designed to overcome clinical inertia for hypertension care did not lead to improvements in blood pressure control. Participant adherence to the intervention was high. The control intervention led to positive changes in the use of preventive services (eg, tetanus immunization) and the intervention condition led to more discussions of hypertension-relevant tests (eg, serum creatinine and urine protein). By providing patients with individually tailored questions to ask during PCP visits, this study demonstrated that participants were likely to discuss the questions with PCPs. These discussions did not, however, lead to improvements in blood pressure control. TRIAL REGISTRATION: ClinicalTrials.gov NCT00377208; http://clinicaltrials.gov/ct2/show/NCT00377208 (Archived by WebCite at http://www.webcitation.org/6IqWiPLon).

The adult ADHD self-report scale for screening for adult attention deficit-hyperactivity disorder (ADHD).

BACKGROUND: Adult attention deficit-hyperactivity disorder (ADHD) is underdiagnosed in the primary care setting despite 3% to 6% of adults having ADHD-like symptoms. The Adult ADHD Self-Report Scale-V1.1 (ASRS-V1.1) is a validated, 6-question screen for adult ADHD. Our purpose was to analyze this tool for evaluating patients in a busy primary care setting. METHODS: The ASRS-V1.1 was administered to patients in 8 busy primary care practices. All with a positive score and a random sample of those with a negative screening score were asked to complete the Conners' Adult ADHD Rating Scale Self Report-Short Version. Each was administered within the clinic setting during the same session. Sensitivity, specificity, and positive/negative predictive values were calculated. Data were evaluated for site-specific differences. RESULTS: It took an average of 54.3 seconds (range, 22-252 seconds) to complete the ASRS-V1.1. There was an inconsistency-adjusted sensitivity of 1.0, a specificity of 0.71, a positive predictive value of 0.52, and a negative predictive value of 1.0. No site-specific differences were found. CONCLUSIONS: Because of its ease of use, short time to administer, high sensitivity, and moderate specificity, the ASRS-V1.1 is an effective adult ADHD screening to guide further evaluations for ADHD.

Profiling retinal biochemistry in the MPDZ mutant retinal dysplasia and degeneration chick: a model of human RP and LCA.

PURPOSE: Raman microscopy, a rapid nondestructive technique that profiles the composition of biological samples, was used to characterize retinal biochemistry in the retinal dysplasia and degeneration (rdd) and wild-type (wt) chick retina during retinogenesis and at hatching. METHODS: Embryonic day (E)13 and posthatch day (P)1 rdd and wt retinal cross-sections (n = 3 of each line at each age) were profiled using 633 helium-neon laser excitation. The biochemical composition was determined using computational analysis of the Raman spectra. In parallel histology, TUNEL and glial fibrillary acidic protein (GFAP) immunostaining were used to visualize retinal dysfunction. RESULTS: Principal component (PC) analysis of the Raman spectra identified 50 major biochemical profiles, but only PCs that made significant contributions to variation within rdd and wt retina were mapped. These significant PCs were shown to arise from DNA, various fatty acids, melanin, and a number of proteins. Distinct patterns of GFAP immunostaining and a larger population of TUNEL-positive nuclei were observed in the rdd versus wt retina. CONCLUSIONS: This study has demonstrated that Raman microscopy can discriminate between major retinal biomolecules, thus providing an unbiased account of how their composition varies due to the impact of the MPDZ null mutation in the rdd chick relative to expression in the normal wt retina.

Mpdz null allele in an avian model of retinal degeneration and mutations in human leber congenital amaurosis and retinitis pigmentosa.

PURPOSE: To identify the defective gene in the sex-linked, recessively inherited retinal dysplasia and degeneration (rdd) chicken and to search for the human equivalent disease. METHODS: Microsatellites from chicken chromosome Z were genotyped in 77 progeny of a carrier male (rdd/+) and an affected female (rdd/W), and candidate genes were sequenced. Retinal cross-sections from rdd and wild-type birds were analyzed by immunohistology. The human orthologous gene was screened in a panel of archival DNAs from 276 patients with retinitis pigmentosa (RP) or Leber congenital amaurosis (LCA) using melting curve analysis and DNA sequencing. RESULTS: The rdd locus was refined to an approximately 3-Mb region on chromosome Z. Sequence analysis identified a C→T change in the mpdz gene that created a premature stop codon (c.1372C→T, p.R458X), which segregated with the disease phenotype. As expected, the full-length mpdz protein was absent in rdd retinas, but in wild-type birds, it localized to the retinal outer limiting membrane, where it may have a role in the interactions between photoreceptors and Müller glia cells. The screen to identify the human equivalent disease found 10 heterozygous variants in the orthologous gene in patients with RP (three missense and two null alleles) and LCA (four missense and one null allele). CONCLUSIONS: These findings reveal that MPDZ is essential for normal development of the retina and may have a role in maintaining photoreceptor integrity. The identification of human mutations suggests that MPDZ plays a role in human retinal disease, but the precise nature of this role remains to be determined.

Academic detailing to increase colorectal cancer screening by primary care practices in Appalachian Pennsylvania.

BACKGROUND: In the United States, colorectal cancer (CRC) is the third most frequently diagnosed cancer and second leading cause of cancer death. Screening is a primary method to prevent CRC, yet screening remains low in the U.S. and particularly in Appalachian Pennsylvania, a largely rural area with high rates of poverty, limited health care access, and increased CRC incidence and mortality rates. Receiving a physician recommendation for CRC screening is a primary predictor for patient adherence with screening guidelines. One strategy to disseminate practice-oriented interventions is academic detailing (AD), a method that transfers knowledge or methods to physicians, nurses or office staff through the visit(s) of a trained educator. The objective of this study was to determine acceptability and feasibility of AD among primary care practices in rural Appalachian Pennsylvania to increase CRC screening. METHODS: A multi-site, practice-based, intervention study with pre- and 6-month post-intervention review of randomly selected medical records, pre- and post-intervention surveys, as well as a post-intervention key informant interview was conducted. The primary outcome was the proportion of patients current with CRC screening recommendations and having received a CRC screening within the past year. Four practices received three separate AD visits to review four different learning modules. RESULTS: We reviewed 323 records pre-intervention and 301 post-intervention. The prevalence of being current with screening recommendation was 56% in the pre-intervention, and 60% in the post-intervention (p=0.29), while the prevalence of having been screened in the past year increased from 17% to 35% (p<0.001). Colonoscopies were the most frequently performed screening test. Provider knowledge was improved and AD was reported to be an acceptable intervention for CRC performance improvement by the practices. CONCLUSIONS: AD appears to be acceptable and feasible for primary care providers in rural Appalachia. A ceiling effect for CRC screening may have been a factor in no change in overall screening rates. While the study was not designed to test the efficacy of AD on CRC screening rates, our evidence suggests that AD is acceptable and may be efficacious in increasing recent CRC screening rates in Appalachian practices which could be tested through a randomized controlled study.

Proteomic profiling of the retinal dysplasia and degeneration chick retina.

PURPOSE: In our previous paper we undertook proteomic analysis of the normal developing chick retina to identify proteins that were differentially expressed during retinal development. In the present paper we use the same proteomic approach to analyze the development and onset of degeneration in the retinal dysplasia and degeneration (rdd) chick. The pathology displayed by the rdd chick resembles that observed in some of the more severe forms of human retinitis pigmentosa. METHODS: Two-dimensional gel electrophoresis (pH 4-7), gel image analysis, and mass spectrometry were used to profile the developing and degenerating retina of the rdd and wild-type (wt) chick retina. RESULTS: Several proteins were identified by mass spectrometry that displayed differential expression between normal and rdd retina between embryonic day 12 (E12) and post-hatch day 1 (P1). Secernin 1 displayed the most significant variation in expression between rdd and wt retina; this may be due to differential phosphorylation in the rdd retina. Secernin 1 has dipeptidase activity and has been demonstrated to play a role in exocytosis; it has been shown to be overexpressed in certain types of cancer and has also been suggested as a potential neurotoxicologically relevant target. Its role in the retina and in particular its differential expression in the degenerate rdd retina remains unknown and will require further investigation. Other proteins that were differentially expressed in the rdd retina included valosin-containing protein, beta-synuclein, stathmin 1, nucleoside diphosphate kinase, histidine triad nucleotide-binding protein, and 40S ribosomal protein S12. These proteins are reported to be involved in several cellular processes, including the ubiquitin proteasome pathway, neuroprotection, metastatic suppression, transcriptional and translational regulation, and regulation of microtubule dynamics. CONCLUSIONS: This proteomic study is the first such investigation of the rdd retina and represents a unique data set that has revealed several proteins that are differentially expressed during retinal degeneration in the rdd chick. Secernin 1 showed the most significant differences in expression during this degeneration period. Further investigation of the proteins identified may provide insight into the complex events underlying retinal degeneration in this animal model.

Protein expression profiling during chick retinal maturation: a proteomics-based approach.

BACKGROUND: The underlying pathways that drive retinal neurogenesis and synaptogenesis are still relatively poorly understood. Protein expression analysis can provide direct insight into these complex developmental processes. The aim of this study was therefore to employ proteomic analysis to study the developing chick retina throughout embryonic (E) development commencing at day 12 through 13, 17, 19 and post-hatch (P) 1 and 33 days. RESULTS: 2D proteomic and mass spectrometric analysis detected an average of 1514 spots per gel with 15 spots demonstrating either modulation or constitutive expression identified via MS. Proteins identified included alpha and beta-tubulin, alpha enolase, B-creatine kinase, gamma-actin, platelet-activating factor (PAF), PREDICTED: similar to TGF-beta interacting protein 1, capping protein (actin filament muscle Z line), nucleophosmin 1 (NPM1), dimethylarginine dimethylaminohydrolase, triosphoaphate isomerase, DJ1, stathmin, fatty acid binding protein 7 (FABP7/B-FABP), beta-synuclein and enhancer of rudimentary homologue. CONCLUSION: This study builds upon previous proteomic investigations of retinal development and represents the addition of a unique data set to those previously reported. Based on reported bioactivity some of the identified proteins are most likely to be important to normal retinal development in the chick. Continued analysis of the dynamic protein populations present at the early stages and throughout retinal development will increase our understanding of the molecular events underpinning retinogenesis.

Tobacco world: evaluation of a tobacco cessation training program for third-year medical students.

BACKGROUND: Training in tobacco cessation counseling is deficient in medical schools. Tobacco World, a tobacco cessation training program, was implemented in a family medicine clerkship and subsequently evaluated. METHOD: In the pilot year, students were assigned to either standard clerkship training (comparison group) or a group that also received Tobacco World training (intervention group). All students received intervention training in the second year of the study. A 35-item questionnaire was administered before and after the four-week clerkship to assess knowledge, attitude, and confidence regarding tobacco cessation counseling. RESULTS: Intervention training was highly rated. Students in the intervention group from both years demonstrated significant improvements in some key measurements of knowledge, attitude, confidence, and increased frequency of tobacco cessation counseling. CONCLUSIONS: This smoking cessation training program addresses an underrepresented area of medical school education and has the potential to translate into improved smoking cessation counseling by future physicians.

Raman microscopy of porcine inner retinal layers from the area centralis.

PURPOSE: To characterize the Raman spectra of porcine inner retinal layers, specifically, the inner nuclear, inner plexiform, ganglion cell, and nerve fiber layers. METHODS: Raman microscopy was employed at three excitation wavelengths, 785, 633, and 514 nm to measure Raman spectra in a high resolution grid across the inner layers of 4% paraformaldehyde cryoprotected porcine retina. Multivariate statistics were used to summarize the principal spectral signals within those layers and to map the distribution of each of those signals. RESULTS: The detected Raman scattering was dominated by a signal characteristic of the protein population present in each layer. As expected, a significant nucleotide contribution was observed in the inner nuclear layer, while the inner plexiform layer displayed a minor contribution from fatty acid based lipid, which would be characteristic of the axonal and synaptic connection resident in this layer. Blood vessels were readily characterized by their distinct heme-derived spectral signature, which increased at 633 and 514 nm excitation compared to 785 nm. Discrete isolated nucleotide signals were identified in the ganglion cell layer, while the nerve fiber layer exhibited a homogenous profile, which is indicative of its broadly uniform axonal and cytoplasmic Muller cell components. CONCLUSIONS: The present study demonstrated the potential of Raman microscopy as a tool to study the biochemical composition of pathologically normal retina. Specifically, the method allowed a unique method of analyzing the network of neurons involved in relaying information from the photoreceptor population to the ganglion cell derived nerve fiber layer. The study has demonstrated the ability of Raman microscopy to generate simultaneously information on a range of specific biochemical entities within the stratified normal retina.

Effect of excitation wavelength on the Raman spectroscopy of the porcine photoreceptor layer from the area centralis.

PURPOSE: Raman microscopy, based upon the inelastic scattering (Raman) of light by molecular species, has been applied as a specific structural probe in a wide range of biomedical samples. The purpose of the present investigation was to assess the potential of the technique for spectral characterization of the porcine outer retina derived from the area centralis, which contains the highest proportion of cone:rod cell ratio in the pig retina. METHODS: Retinal cross-sections, immersion-fixed in 4% (w/v) PFA and cryoprotected, were placed on salinized slides and air-dried prior to direct Raman microscopic analysis at three excitation wavelengths, 785 nm, 633 nm, and 514 nm. RESULTS: Raman spectra of each of the photoreceptor inner and outer segments (PIS, POS) and of the outer nuclear layer (ONL) of the retina acquired at 785 nm were dominated by vibrational features characteristic of proteins and lipids. There was a clear difference between the inner and outer domains in the spectroscopic regions, amide I and III, known to be sensitive to protein conformation. The spectra recorded with 633 nm excitation mirrored those observed at 785 nm excitation for the amide I region, but with an additional pattern of bands in the spectra of the PIS region, attributed to cytochrome c. The same features were even more enhanced in spectra recorded with 514 nm excitation. A significant nucleotide contribution was observed in the spectra recorded for the ONL at all three excitation wavelengths. A Raman map was constructed of the major spectral components found in the retinal outer segments, as predicted by principal component analysis of the data acquired using 633 nm excitation. Comparison of the Raman map with its histological counterpart revealed a strong correlation between the two images. CONCLUSIONS: It has been demonstrated that Raman spectroscopy offers a unique insight into the biochemical composition of the light-sensing cells of the retina following the application of standard histological protocols. The present study points to the considerable promise of Raman microscopy as a component-specific probe of retinal tissue.

Preparation of planar retinal specimens: verification by histology, mRNA profiling, and proteome analysis.

PURPOSE: Elucidation of the transcriptome and proteome of the normal retina will be difficult since it is comprised of at least 55 different cell types. However the characteristic layered cellular anatomy of the retina makes it amenable to planar sectioning, enabling the generation of enriched retinal cell populations. The aim of this study was to validate a reproducible method for preparing enriched retinal layers from porcine retina. METHODS: The thicknesses of the retinal photoreceptor, inner nuclear and ganglion cell, and fiber layers were determined by routine histology of cross sections of fresh whole retina mounted on polyvinylidene difluoride (PVDF) membrane. Dissected retina (5 mm2) was placed on PVDF membrane and a series of planar cryosections corresponding to the photoreceptor and inner nuclear layer were removed leaving the ganglion cell and fiber layer which was subsequently detached from the membrane. The retinal specimens were stored at -80 degrees C. Representative planar tissue sections were sonicated in ice-chilled 40 mM ammonium bicarbonate pH 7.9 and aliquots removed for RNA extraction. Quantitative RT-PCR was used to analyze the mRNA expression of genes indicative of specific retinal layers. Ammonium bicarbonate protein extracts were centrifuged, lyophilized and prepared for direct liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis using a Waters Q-Tof Ultima. RESULTS: Histological analysis established the parameters for planar cryosectioning: photoreceptor layer (69+/-1.8 microm), outer plexiform (11+/-0.6 microm), inner nuclear layer (28+/-0.5 microm), inner plexiform, ganglion cell and fiber layer (100+/-5.3 microm). Gene expression profiling provided an independent method for validating the respective retinal preparations. For example, glial fibrillary acidic protein (GFAP) was expressed up to 21 fold higher in the inner retinal "ganglion cell enriched" fraction than in the outer retinal "photoreceptor enriched" fraction. The pattern was reversed for blue cone opsin, which was expressed up to 24 fold higher in the "photoreceptor enriched" fraction. Endogenous protein fragments indicative of each layer were identified by mass spectrometry and de novo sequence data obtained. CONCLUSIONS: Combined histological and mRNA expression profiling has confirmed the development of a reproducible method for generating validated porcine retinal layers enriched for specific cell types. Direct proteome analysis detected endogenous peptide fragments of characteristic retinal proteins. Further analysis of these enriched retinal cell preparations will facilitate a more selective investigation of the retinal transcriptome and proteome than studies of the intact retina.