Jan Schroeter

Temporal Retinal Nerve Fiber Loss in Patients with Spinocerebellar Ataxia Type 1

Background Autosomal dominant spinocerebellar ataxia type 1 is an adult onset progressive disorder with well characterized neurodegeneration in the cerebellum and brainstem. Beyond brain atrophy, few data exist concerning retinal and optic nerve involvement. Objective To evaluate retinal changes in SCA1 patients compared to age and gender matched healthy controls. Methodology/Principal Findings Nine patients with SCA1 were prospectively recruited from the ataxia clinic and were compared to nine age and gender matched healthy controls. Both cohorts received assessment of visually evoked potentials and eye examination by optical coherence tomography to determine retinal nerve fiber layer thickness and total macular volume. While no differences were found in visually evoked potentials, SCA1 patients showed a significant reduction of mean retinal nerve fiber layer thickness (RNFLT) compared to healthy controls (84±13 µm vs. 97±8 µm, p = 0.004). Temporal areas showed the most prominent RNFLT reduction with high statistical significances (temporal-inferior: p<0.001, temporal: p<0.001, temporal-superior: p = 0.005) whereas RNFLT in nasal areas was in the range of the control group. From six SCA1 patients an additional macular scan was obtained. The comparison to the corresponding healthy control showed a slight but not significant reduction in TMV (8.22±0.68 mm3 vs. 8.61±0.41 mm3, p = 0.15). Conclusion In SCA1 patients, we found evidence for degeneration of retinal nerve fibers. The temporal focus of the observed retinal nerve fiber layer reduction suggests an involvement of the papillo-macular bundle which resembles pathology found in toxic or mitochondrial optic nerve disease such as Lebers hereditary optic neuropathy (LHON) or dominant optic atrophy (DOA).

Evaluation of risk factors for retinal damage due to chloroquine and hydroxychloroquine

Background/Aims To evaluate risk factors for retinal damage due to the intake of chloroquine and hydroxychloroquine. Methods In a retrospective chart review, patients receiving or having received one of the drugs were classified as affected by maculopathy or retinopathy, or as not affected on the basis of the documented findings. Uncertain cases were excluded. The risk factors as postulated by the American Academy of Ophthalmology (AAO) and additional factors like diagnosis of underlying disease, total dose, nicotine abuse and the sum of the AAO risk factors were compared between both groups. Results 51 patients with a history of or ongoing treatment with chloroquine (23 individuals) or hydroxychloroquine (28 individuals) were included. Most of the postulated risk factors were expectedly elevated in the affected group. Significant differences applied to age, duration of intake and the sum of AAO risk factors. Surprisingly, positive smoking history was more frequent in the not affected. The toxic threshold of the daily chloroquine dose was exceeded by most of the patients. Conclusions Age and the duration of intake are major risk factors. Smoking seems to be negligible. The sum of AAO risk factors can give an estimation of the individual risk profile. Individual and weight-adapted dosing is especially essential for chloroquine.

Corneal trephination with the femtosecond laser.

Purpose: To evaluate the feasibility and cut quality of corneal trephination in human donor corneal tissue with the femtosecond laser. Methods: Twelve human corneoscleral discs were inserted in an artificial anterior chamber. After corneal thickness measurement and tonometry, the cornea was mounted on a femtosecond laser (FEMTEC; 20/10 Perfect Vision, Heidelberg, Germany) through a contact lens (patient interface). Trephination was performed with diameters of 7.0, 7.5, 8.0, and 8.5 mm in 3 corneas each. The corneal button was removed from the corneoscleral disc in 2 of the 3 corneas in each case. The cut was not manipulated in the remaining corneas to enable histologic detection of possible tissue bridges. The cut edges were macroscopically and light-microscopically examined for quality. Results: Corneal buttons and corneoscleral discs could be separated by blunt dissection in all cases. Tissue bridges were more common in thicker edematous corneas than in thinner ones. Both the macro- and microscopic examination disclosed smooth rectilinear cut margins with a perpendicular cut edge. Conclusion: This feasibility study shows that the femtosecond laser enables sufficient trephination of human donor corneas.

[Chloroquine/hydroxychloroquine: variability of retinotoxic cumulative doses].

OBJECTIVE The critical dose of chloroquine/hydroxychloroquine leading to a maculopathy or generalised retinopathy remains undetermined. In the literature, 100 g is considered the dose at which regular vision checks should be performed. Generally, chloroquine is said to be more toxic than hydroxychloroquine. A young patient presenting with toxic maculopathy after 57 g of hydroxychloroquine and a daily dosage of 2 mg/kg body weight prompted us to retrospectively look at our patients examined in this respect over about 1 year. METHODS The data of patients who were examined because of chloroquine/hydroxychloroquine intake or a respective maculopathy/retinopathy were retrospectively analysed. The time period was January 2005 until March 2006. Retinal damage was defined by fundus changes and alteration of the multifocal electroretinogram (ERG). RESULTS Twenty-one patients--18 women and three men--were examined. The mean age was 51 years (range 6-71). Five of the nine chloroquine-treated patients developed a maculopathy, and one of them developed an additional generalised retinopathy. Of the patients treated by hydroxychloroquine, three of 12 suffered from a maculopathy and one from an additional generalised retinopathy. The cumulative doses leading to retinal damage ranged from 170 g to 1650 g for chloroquine and from 57 g to 1190 g for hydroxychloroquine. The highest cumulative doses without leading to signs of retinopathy were 790 g for chloroquine and 1200 g for hydroxychloroquine. CONCLUSIONS There is a high variability of cumulative doses of chloroquine/hydroxychloroquine that lead to a toxic retinopathy. Therefore, early and regular ophthalmologic examinations are recommended. Electrophysiological testing should be performed once a year, corresponding to about 60 g of base with one tablet a day. For electrophysiology, the multifocal ERG has turned out to be the most important test in this regard. However, visual acuity and funduscopy should be performed more frequently.

A system for the automatic estimation of morphometric parameters of corneal endothelium in alizarine red stained images

Background/aims A computer program for the automatic estimation of endothelium morphometric parameters (cell density, pleomorphism, polymegethism) in alizarine red-stained images is presented and evaluated. Methods Images of corneal endothelium from 30 porcine eyes stained with alizarine red were acquired with an optical microscope and saved as grey-level digital images. Each image was first pre-processed for luminosity correction and contrast enhancement. An artificial neural network was used to classify all pixels as cell contour or cell body pixels. The segmented cell contours were then used to obtain estimates of morphometric parameters. The central area was assessed and the mean area per cornea was 0.54±0.07 mm2. The whole system was implemented as a computer program using the Matlab language. Estimated parameters were compared with the corresponding values derived from manual contour detection on the same images used for the automatic estimation. Results For the 30 images in our dataset, the mean differences for automatic versus manual parameters were −12±52 (range −103 to +145) cells/mm2 for density, 0.5±2.6% (range −5.6 to +5.6%) for pleomorphism and −0.7±1.9% (range −4.1 to +2.8%) for polymegethism. Conclusion The evaluation of the automatic system on 30 images from porcine eyes confirmed its ability to estimate reliably morphometric parameters with respect to parameter values derived by manual analysis.

Endothelial evaluation in the cornea bank.

The light microscope is the first-choice technique for the evaluation of organ-cultured donor corneas. For the microscopic visualization of the endothelial cells, the corneas have to be immersed in ahypotonic solution. The number of cells, their vitality and morphology are analyzed. The cell densityis easily estimated with the fixed frame/L method. Use of an image analysis system enables computer processing and counting of digitalized endothelial cell images. An adequately high endothelial cell density after the culture period is a decisive criterion that must be met before releasing adonor cornea for grafting. An endothelial cell density of 2,000-2,200 cells/mm2 is generally recognized as the lower limit for the longest possible graft survival. While an evaluation of the endothelium does not necessarily have to be performed at the beginning or middle of organ culture, it isobligatory at the end. Morphology assessment should routinely involve estimating the pleomorphism (deviation from hexagonality), polymegathism (variation in cell area) and granulation/vacuolization of the endothelial cells. Recognition of devitalized cells is easily facilitated by vital stainingwith trypan blue.

Variabilität der retinotoxischen Gesamtdosis Chloroquin/Hydroxychloroquin

OBJECTIVE The critical dose of chloroquine/hydroxychloroquine leading to a maculopathy or generalised retinopathy remains undetermined. In the literature, 100 g is considered the dose at which regular vision checks should be performed. Generally, chloroquine is said to be more toxic than hydroxychloroquine. A young patient presenting with toxic maculopathy after 57 g of hydroxychloroquine and a daily dosage of 2 mg/kg body weight prompted us to retrospectively look at our patients examined in this respect over about 1 year. METHODS The data of patients who were examined because of chloroquine/hydroxychloroquine intake or a respective maculopathy/retinopathy were retrospectively analysed. The time period was January 2005 until March 2006. Retinal damage was defined by fundus changes and alteration of the multifocal electroretinogram (ERG). RESULTS Twenty-one patients--18 women and three men--were examined. The mean age was 51 years (range 6-71). Five of the nine chloroquine-treated patients developed a maculopathy, and one of them developed an additional generalised retinopathy. Of the patients treated by hydroxychloroquine, three of 12 suffered from a maculopathy and one from an additional generalised retinopathy. The cumulative doses leading to retinal damage ranged from 170 g to 1650 g for chloroquine and from 57 g to 1190 g for hydroxychloroquine. The highest cumulative doses without leading to signs of retinopathy were 790 g for chloroquine and 1200 g for hydroxychloroquine. CONCLUSIONS There is a high variability of cumulative doses of chloroquine/hydroxychloroquine that lead to a toxic retinopathy. Therefore, early and regular ophthalmologic examinations are recommended. Electrophysiological testing should be performed once a year, corresponding to about 60 g of base with one tablet a day. For electrophysiology, the multifocal ERG has turned out to be the most important test in this regard. However, visual acuity and funduscopy should be performed more frequently.

Validation of the Microbiological Testing of Tissue Preparations Using the BACTEC™ Blood Culture System

Background: Since blood culture bottles are validated by the manufacturer for blood only, an additional validation for the use with fluids of tissue preparations is necessary. Methods: Two 10-ml samples of cornea culture medium, histidine-tryptophan-ketoglutarate (HTK) solution, or Ringer solution at the end of femur head thermo-disinfection were given into blood culture bottles (BD BACTECTM Plus Aerobic/F, Anaerobic/F for cornea culture medium and BD BACTECTM Standard Aerobic/ Anaerobic for HTK and Ringer solution) and subsequently spiked with 10–100 colony forming units (CFU) of bacteria or fungi (aerobic bacteria: Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa; anaerobic bacteria: Clostridium sporogenes; fungi: Candida albicans, Aspergillus brasiliensis) according to the European Pharmacopoeia Chapter 2.6.1. Results: All tested bacteria and fungi could be detected in all solutions. All positive and negative controls were tested correctly. Compared to the positive controls, the microbial growth was delayed in the antibiotic-containing cornea culture medium, and negative in two cases of B. subtilis spiking. Conclusion: The use of BACTECTM blood culture bottles seems to be a suitable method for microbiological testing of HTK solution, Ringer solution, and, with limitations, also for testing of the antibiotic-containing cornea culture medium.

Procedural guidelines. Good tissue practice for cornea banks

A cornea bank must have an organizational structure in which responsibility and authority to issue directives are clearly defined. It must also use a documented quality management system on the basis of good practice procedures which is maintained to the current standards. The personnel of a cornea bank must be present in sufficient numbers and be suitably qualified. A cornea bank must be in possession of appropriate facilities which are suitable for the main purpose of conservation of donor corneas. All equipment must be designed and maintained corresponding to the intended purpose. Deviations from the stipulated quality and safety standards must give rise to documented investigations which include decisions on options for correctional and preventive measures. Acquisition of donors and tissue sampling must be strictly controlled and documented. This also applies to entry of donor tissue in the cornea bank. During conservation a microscopic examination of the endothelial cell layer must be carried out at least once. Measures must be taken to keep the risk of contamination as low as possible. Donor corneal tissue can only be released if defined criteria are fulfilled. Any suspicion of severe undesired reactions and events for the recipient of a corneal transplant must be registered with the authorities. The activities of a cornea bank must maintain and adapt the state-of-the-art with respect to scientific progress.

Augenhornhaut – Banken und klinische Anwendung

Das neue Gewebegesetz als Folge der Umsetzung der Richtlinien des EU-Parlaments und des Rates 2004/23/EG, 2006/17/EG und 2006/86/EG wird an den Prozessen in der Hornhautbank nichts grundlegend ändern. Es ist aber eine zunehmende Transparenz bei der Gewinnung und Prozessierung von Spenderhornhäuten, eine Harmonisierung bestimmter Arbeitsschritte und ein erhöhtes Bewusstsein für die Bedeutung validierter Abläufe zu erwarten. Bei der Umsetzung des Gesetzes müssen die Besonderheiten der humanen Augenhornhaut berücksichtigt werden, insbesondere ihre natürliche Unsterilität, fehlende Sterilisierbarkeit, postmortale Explantationszeit bis zu 72 h und ihre Transplantierbarkeit trotz Karzinomerkrankung und bakterieller Sepsis des Spenders. Der Tätigkeitsschwerpunkt der Hornhautbanken bleibt die Bereitstellung von Hornhauttransplantaten mit normaler physiologischer Funktionalität und minimalem Risiko einer Krankheitsübertragung. Die Steigerung der Anzahl transplantierbarer Spenderhornhäute ist zur Überwindung des weiterhin bestehenden Mangels dabei eine wesentliche Aufgabe.