Contrast Sensitivity Mediated by Inferred Magno- and Parvocellular Pathways in Type 2 Diabetics with and without Nonproliferative Retinopathy

PURPOSE. To evaluate achromatic contrast sensitivity (CS) with magnocellular-(M) and parvocellular-(P) probing stimuli in type 2 diabetics, with (DR) or without (NDR) nonproliferative retinopathy. METHODS. Inferred M-and P-dominated responses were assessed with a modified version of the steady-/pulsed-pedestal paradigm (SP/PP) applied in 26 NDR (11 male; mean age, 55 +/- 9 years; disease duration, 5 +/- 4 years); 19 DR (6 male; mean age, 58 +/- 7 years; disease duration = 9 +/- 6 years); and 18 controls (CTRL; 12 male; mean age, 55 +/- 10 years). Thresholds were measured with pedestals at 7, 12, and 19 cd/m(2), and increment durations of 17 and 133 ms. The thresholds from the two stimulus durations were used to estimate critical durations (Tc) for each data set. RESULTS. Both DR and NDR patients had significant reduction in CS in both SP and PP paradigms in relation to CTRL (Kruskal-Wallis, P < 0.01). Patients` critical duration estimates for either paradigm were not significantly different from CTRL. CONCLUSIONS. The significant reduction of CS in both paradigms is consistent with losses of CS in both M and P pathways. The CS losses were not accompanied by losses in temporal processing speed in either diabetic group. Significant CS loss in the group without retinopathy reinforces the notion that neural changes associated with the cellular and functional visual loss may play an important role in the etiology of diabetic visual impairment. In addition, the results show that the SP/PP paradigm provides an additional tool for detection and characterization of the early functional damage due to diabetes. (Invest Ophthalmol Vis Sci. 2011; 52:1151-1155) DOI:10.1167/iovs.09-3705

Phosphene Thresholds Elicited by Transcorneal Electrical Stimulation in Healthy Subjects and Patients with Retinal Diseases

PURPOSE. To evaluate electrically evoked phosphene thresholds (EPTs) in healthy subjects and in patients with retinal disease and to assess repeatability and possible correlations with common ophthalmologic tests. METHODS. In all, 117 individuals participated: healthy subjects (n = 20) and patients with retinitis pigmentosa (RP, n = 30), Stargardt's disease (STG, n = 14), retinal artery occlusion (RAO, n = 20), nonarteritic anterior ischemic optic neuropathy (NAION, n = 16), and primary open-angle glaucoma (POAG, n = 17). EPTs were determined at 3, 6, 9, 20, 40, 60, and 80 Hz with 5+5-ms biphasic current pulses using DTL electrodes. Subjects were examined twice (test-retest range: 1-6 weeks). An empirical model was developed to describe the current-frequency relationship of EPTs. Visual acuity, visual field (kinetic + static), electrophysiology (RP, RAO, STG: Ganzfeld-electroretinography [ERG]/multifocal-ERG; POAG: pattern-ERG; NAION: VEP), slit-lamp biomicroscopy, fundus examination, and tonometry were assessed. RESULTS. EPTs varied between disease groups (20 Hz: healthy subjects: 0.062 +/- 0.038 mA; STG: 0.102 +/- 0.097 mA; POAG: 0.127 +/- 0.09 mA; NAION: 0.244 +/- 0.126 mA; RP: 0.371 +/- 0.223 mA; RAO: 0.988 +/- 1.142 mA). In all groups EPTs were lowest at 20 Hz. In patients with retinal diseases and across all frequencies EPTs were significantly higher than those in healthy subjects, except in STG at 20 Hz (P = 0.09) and 40 Hz (P = 0.17). Test-retest difference at 20 Hz was 0.006 mA in the healthy group and 0.003-0.04 mA in disease groups. CONCLUSIONS. Considering the fast, safe, and reliable practicability of EPT testing, this test might be used more often under clinical circumstances. Determination of EPTs could be potentially useful in elucidation of the progress of ophthalmologic diseases, either in addition to standard clinical assessment or under conditions in which these standard tests cannot be used meaningfully. (ClinicalTrials.gov number, NCT00804102.) (Invest Ophthalmol Vis Sci. 2012; 53: 7440-7448) DOI:10.1167/iovs.12-9612

Transcorneal Electrical Stimulation Shows Neuroprotective Effects in Retinas of Light-Exposed Rats

PURPOSE. To examine the effects of transcorneal electrical stimulation (TES) on retinal degeneration of light-exposed rats. METHODS. Thirty-three Sprague Dawley albino rats were divided into three groups: STIM (n = 15) received 60 minutes of TES, whereas SHAM (n = 15) received identical sham stimulation 2 hours before exposure to bright light with 16,000 lux; healthy animals (n = 3) served as controls for histology. At baseline and weekly for 3 consecutive weeks, dark-and light-adapted electroretinography was used to assess retinal function. Analysis of the response versus luminance function retrieved the parameters Vmax (saturation amplitude) and k (luminance to reach 1/2Vmax). Retinal morphology was assessed by histology (hematoxylin-eosin [HE] staining; TUNEL assay) and immunohistochemistry (rhodopsin staining). RESULTS. Vmax was higher in the STIM group compared with SHAM 1 week after light damage (mean intra-individual difference between groups 116.06 mu V; P = 0.046). The b-wave implicit time for the rod response (0.01 cd.s/m(2)) was lower in the STIM group compared with the SHAM group 2 weeks after light damage (mean intra-individual difference between groups 5.78 ms; P = 0.023); no other significant differences were found. Histological analyses showed photoreceptor cell death (TUNEL and HE) in SHAM, most pronounced in the superior hemiretina. STIM showed complete outer nuclear layer thickness preservation, reduced photoreceptor cell death, and preserved outer segment length compared with SHAM (HE and rhodopsin). CONCLUSIONS. This sham-controlled study shows that TES can protect retinal cells against mild light-induced degeneration in Sprague Dawley rats. These findings could help to establish TES as a treatment in human forms of retinal degenerative disease. (Invest Ophthalmol Vis Sci. 2012;53:5552-5561) DOI: 10.1167/iovs.12-10037

Retinal projections and neurochemical characterization of the pregeniculate nucleus of the common marmoset (Callithrix jacchus)

In mammals, the suprachiasmatic nucleus (SCN) and the intergeniculate leaflet (IGL) are the main components of the circadian timing system. The SCN is the site of the endogenous biological clock that generates rhythms and synchronizes them to environmental cues. The IGL is a key structure that modulates SCN activity and is responsible for the transmission of non-photic information to the SCN, thus participating in the integration between photic and non-photic stimuli. Both the SCN and IGL receive projections of retinal ganglion cells and the IGL is connected to the SCN through the geniculohypothalamic tract. Little is known about these structures in the primate brain and the pregeniculate nucleus (PGN) has been suggested to be the primate equivalent of the rodent IGL. The aim of this study was to characterize the PGN of a primate, the common marmoset (Callithrix jacchus), and to analyze its retinal afferents. Here, the marmoset PGN was found to be organized into three subsectors based on neuronal size, pattern of retinal projections, and the distribution of neuropeptide Y-, GAD-, serotonin-, enkephalin- and substance P-labeled terminals. This pattern indicates that the marmoset PGN is equivalent to the IGL. This detailed description contributes to the understanding of the circadian timing system in this primate species considering the importance of the IGL within the context of circadian regulation. (C) 2012 Elsevier B.V. All rights reserved.

Retinal cell death induced by TRPV1 activation involves NMDA signaling and upregulation of nitric oxide synthases

The activation of the transient receptor potential vanilloid type 1 channel (TRPV1) has been correlated with oxidative and nitrosative stress and cell death in the nervous system. Our previous results indicate that TRPV1 activation in the adult retina can lead to constitutive and inducible nitric oxide synthase-dependent protein nitration and apoptosis. In this report, we have investigated the potential effects of TRPV1 channel activation on nitric oxide synthase (NOS) expression and function, and the putative participation of ionotropic glutamate receptors in retinal TRPV1-induced protein nitration, lipid peroxidation, and DNA fragmentation. Intravitreal injections of the classical TRPV1 agonist capsaicin up-regulated the protein expression of the inducible and endothelial NOS isoforms. Using 4,5-diaminofluorescein diacetate for nitric oxide (NO) imaging, we found that capsaicin also increased the production of NO in retinal blood vessels. Processes and perikarya of TRPV1-expressing neurons in the inner nuclear layer of the retina were found in the vicinity of nNOS-positive neurons, but those two proteins did not colocalize. Retinal explants exposed to capsaicin presented high protein nitration, lipid peroxidation, and cell death, which were observed in the inner nuclear and plexiform layers and in ganglion cells. This effect was partially blocked by AP-5, a NMDA glutamate receptor antagonist, but not by CNQX, an AMPA/kainate receptor antagonist. These data support a potential role for TRPV1 channels in physiopathological retinal processes mediated by NO, which at least in part involve glutamate release.

Mitochondrial dysfunction in hereditary optic neuropathies

MITOCHONDRIAL DYSFUNCTION IN HEREDITARY OPTIC NEUROPATHIES Mitochondrial pathologies are a heterogeneous group of clinical manifestations characterized by oxidative phosphorylation impairment. At the beginning of their recognition mitochondrial pathologies were regarded as rare disorders but indeed they are more frequent than originally thought. Due to the unique mitochondria peculiarities mitochondrial pathologies can be caused by mutations in both mitochondrial and nuclear genomes. The poor knowledge of pathologic mechanism of these disorders has not allowed a real development of the “mitochondrial medicine”, that is currently limited to symptoms mitigation. Leber hereditary optic neuropathy (LHON) was the first pathology to be linked to a point mutation in the mtDNA. The mechanism by which point mutations in mitochondrial gene encoding Complex I subunits leads to optic nerve degeneration is still unknown, although is well accepted that other genetic or environmental factors are involved in the modulation of pathology, where a pivotal role is certainly played by oxidative stress. We studied the relationship between the Ala16Val dimorphism in the mitochondrial targeting sequence of nuclear gene SOD2 and the 3460/ND1 LHON mutation. Our results show that, in control population, the heterozygous SOD2 genotype is associated to a higher activity and quantity of MnSOD, particularly with respect to Val homozygotes. Furthermore, we demonstrated that LHON patients harboring at least one Ala allele are characterized by an increased MnSOD activity with respect to relative control population. Since the ATP synthesis rate – severely reduced in LHON patients lymphocytes - is not affected by the SOD2 genotype, we concluded that SOD2 gene could modulate the pathogenicity of LHON mutations through a mechanism associated to an increase of reactive oxygen species production. Autosomal dominant optic atrophy (ADOA) is a pathology linked to mutations in nuclear gene encoding Opa1, a dynamin-related protein localized in the mitochondrial matrix. Although the clinical course is slightly different, the endpoint of ADOA is exactly the same of LHON: optic nerve degeneration with specific involvement of retinal ganglion cells. Opa1 is a relatively new protein, whose major role is the regulation of mitochondrial fusion. Mitochondrial morphology is the results of the equilibrium between two opposite force: fusion and fission, two processes that have to be finely regulated in order to preserve mitochondrial and cellular physiology. We studied fibroblasts deriving from ADOA patients characterized by a new deletion in the GTPase domain of the OPA1 gene. The biochemical characterization of ADOA and control fibroblasts has concerned the evaluation of ATP synthesis rate, mitochondrial membrane potential in different metabolic conditions and the morphological status of mitochondria. Regarding ATP synthesis rate we did not find significant differences between ADOA and control fibroblasts even though a trend toward increased reduction in ADOA samples is observed when fibroblasts are grown in absence of glucose or in the medium containing gramicidin. Furthermore, we found that also in ADOA fibroblasts membrane potential is actively maintained by proton pumping of fully functional respiratory chain complexes. Our results indicate that the mutation found in the pedigree analyzed acts primary impairing the mitochondrial fusion without affecting the energy production, supporting the notion that cell function is tightly linked to mitochondrial morphology. Mitochondrial dysfunctions are acquiring great attention because of their recognized relevance not only in aging but also in age-related pathologies including cancer, cardiovascular disease, type II diabetes, and neurodegenerative disorders. The involvement of mitochondria in such detrimental pathologies that, currently, have become so common enhances the necessity of standardization of therapeutic strategies capable of rescuing the normal mitochondrial function. In order to propose an alternative treatment for energy deficiency-disorders we tested the effect of substrates capable to stimulate the substrate-level phosphorylation on viability and energy availability in different experimental models grown under different metabolic conditions. In fibroblasts, the energy defect was achieved by culturing cells in presence of oligomycin, an inhibitor of ATP synthase complex. NARP cybrids have been used as model of mitochondrial pathology. Cell viability and ATP content have been considered as parameters to assay the capability of exogenous substrate to rescue energy failure. Our results suggest that patients suffering for some forms of ATP synthase deficiency, or characterized by a deficiency in energy production, might benefit from dietary or pharmacological treatment based on supplementation of α-ketoglutarate and aspartate.

Molecular bases, pathogenic mechanisms and possible therapeutic approach in Leber's Hereditary Optic Neuropathy

Leber’s hereditary optic neuropathy (LHON) is a mitochondrial disease characterized by a rapid loss of central vision and optic atrophy, due to the selective degeneration of retinal ganglion cells. The age of onset is around 20, and the degenerative process is fast and usually the second eye becomes affected in weeks or months. Even if this pathology is well known and has been well characterized, there are still open questions on its pathophysiology, such as the male prevalence, the incomplete penetrance and the tissue selectivity. This maternally inherited disease is caused by mutations in mitochondrial encoded genes of NADH ubiquinone oxidoreductase (complex I) of the respiratory chain. The 90% of LHON cases are caused by one of the three common mitochondrial DNA mutations (11778/ND4, 14484/ND6 and 3460/ND1) and the remaining 10% is caused by rare pathogenic mutations, reported in literature in one or few families. Moreover, there is also a small subset of patients reported with new putative pathogenic nucleotide changes, which awaits to be confirmed. We here clarify some molecular aspects of LHON, mainly the incomplete penetrance and the role of rare mtDNA mutations or variants on LHON expression, and attempt a possible therapeutic approach using the cybrids cell model. We generated novel structural models for mitochondrial encoded complex I subunits and a conservation analysis and pathogenicity prediction have been carried out for LHON reported mutations. This in-silico approach allowed us to locate LHON pathogenic mutations in defined and conserved protein domains and can be a useful tool in the analysis of novel mtDNA variants with unclear pathogenic/functional role. Four rare LHON pathogenic mutations have been identified, confirming that the ND1 and ND6 genes are mutational hot spots for LHON. All mutations were previously described at least once and we validated their pathogenic role, suggesting the need for their screening in LHON diagnostic protocols. Two novel mtDNA variants with a possible pathogenic role have been also identified in two independent branches of a large pedigree. Functional studies are necessary to define their contribution to LHON in this family. It also been demonstrated that the combination of mtDNA rare polymorphic variants is relevant in determining the maternal recurrence of myoclonus in unrelated LHON pedigrees. Thus, we suggest that particular mtDNA backgrounds and /or the presence of specific rare mutations may increase the pathogenic potential of the primary LHON mutations, thereby giving rise to the extraocular clinical features characteristic of the LHON “plus” phenotype. We identified the first molecular parameter that clearly discriminates LHON affected individuals from asymptomatic carriers, the mtDNA copy number. This provides a valuable mechanism for future investigations on variable penetrance in LHON. However, the increased mtDNA content in LHON individuals was not correlated to the functional polymorphism G1444A of PGC-1 alpha, the master regulator of mitochondrial biogenesis, but may be due to gene expression of genes involved in this signaling pathway, such as PGC-1 alpha/beta and Tfam. Future studies will be necessary to identify the biochemical effects of rare pathogenic mutations and to validate the novel candidate mutations here described, in terms of cellular bioenergetic characterization of these variants. Moreover, we were not able to induce mitochondrial biogenesis in cybrids cell lines using bezafibrate. However, other cell line models are available, such as fibroblasts harboring LHON mutations, or other approaches can be used to trigger the mitochondrial biogenesis.

Elektrophysiologische Untersuchungen am experimentellen Autoimmun-Glaukom-Modell

Das Glaukom ist eine der führenden Erblindungsursachen weltweit. Trotzdem ist die Pathogenese, die zur Degeneration der retinalen Ganglienzellen führt, bisher nicht verstanden. In den letzten Jahren ergaben sich verschiedene Hinweise auf die Beteiligung einer immunologischen Komponente. Thema dieser Arbeit waren elektrophysiologische Untersuchungen, im Sinne von visuell evozierten Potentialen, am Tiermodell des Experimentellen Autoimmun Glaukoms und die Etablierung dieses Modells. Das Modell basiert auf einer Immunisierung von Lewisratten mit Pertussistoxin, inkompletten Freunds Adjuvant und potentiellen Antigenen, die zu einer Immunreaktion und einem Verlust von retinalen Ganglienzellen führen sollen. Zur Etablierung des Experimentellen Autoimmun Glaukom Modells wurde eine fünfwöchige Studie mit vier Gruppen durchgeführt. Als Antigene wurden Glia fibrilläres saures Protein (n= 10) und Myelin basisches Protein (n=10) verwendet, die beide in Studien zu Serum- und Kammerwasseranalysen bei Glaukompatienten eine Abweichung zur Kontrollgruppe gezeigt hatten. Außerdem wurde eine Gruppe mit selbst hergestelltem Sehnerv-Homogenat (n=12) immunisiert. Eine Gruppe erhielt keine Immunisierung und diente als Kontrolle (n=10). Zur Überprüfung der Effekte des Modells dienten verschiedene Untersuchungsmethoden, wie die Augeninnendruckmessung und die Untersuchung der Fundi. Des Weiteren wurden transiente und stationäre visuell evozierte Potentiale abgeleitet und die Latenzen, Amplituden und die Marker S (Steigung) und TR (Temporale Antworten) verglichen. Außerdem erfolgte nach Tötung der Tiere die Entnahme der Gehirne und Augen. Die Gehirne wurden nach Paraffineinbettung geschnitten, mit Luxol Fast Blue und Kresylviolett gefärbt und hinsichtlich etwaiger Entmarkungsherde oder anderer Pathologien unter dem Mikroskop bewertet. Der Verlauf des intraokulären Drucks zeigte sowohl zwischen den Gruppen als auch zwischen den verschiedenen Zeitpunkten keine signifikanten Unterschiede. Er bewegte sich im physiologischen Bereich mit durchschnittlich circa 12 mmHg. Die Funduskopien lieferten zu keinem Zeitpunkt krankhafte Veränderungen. Auch die visuell evozierten Potentiale lieferten zwischen den Gruppen keine signifikanten Unterschiede, sondern belegten normale visuelle Funktion bei allen Tieren. Die Auswertung der histologischen Untersuchung der Hirnschnitte zeigte keine Entmarkungsherde. Die erzielten Ergebnisse dieser Arbeit legen nahe, dass der retinale Ganglienzellverlust beim Experimentellen Autoimmun Glaukom Modell ohne eine Augeninnendruckerhöhung stattfindet. Die Fundusuntersuchung und die visuell evozierten Potentiale, wie in diesem Versuchsaufbau durchgeführt, scheinen nicht sensibel genug zu sein, diese Verluste nachzuweisen. In weiteren Arbeiten sollten andere Methoden zum Nachweis der retinalen Ganglienzellverluste erprobt werden. Neben elektrophysiologischen Methoden bieten sich für das weitere Vorgehen besonders immunhistologische Methoden an. Außerdem sollten die Mechanismen erforscht werden durch die es nach der Immunisierung zur Apoptose von retinalen Ganglienzellen kommt und welche Antikörper dazuführen können. Des Weiteren ist von Interesse, ob und wie eine zelluläre Komponente an der Pathogenese des Experimentellen Autoimmun Glaukoms beteiligt ist.

Analyse immunologischer und proteomischer Aspekte im Rahmen der Glaukompathogenese

Das Glaukom stellt eine heterogene Gruppe von okularen Erkrankungen dar, deren Pathogenese sich durch einen langsamen, progradienten Untergang von retinalen Ganglienzellen und ihren Axonen auszeichnet. rnIn den letzten Jahren wurde im Kontext der Glaukompathogenese verstärkt die Beteiligung autoreaktiver Antikörper diskutiert. Ein Schwerpunkt dieser Arbeit bestand in dem Vergleich solcher Autoantikörper-Reaktionen in den Serum- und Kammerwasserproben einzelner Glaukompatienten. Hierdurch sollte geklärt werden, inwieweit die Immunreaktivitäten dieser beiden Körperflüssigkeiten miteinander übereinstimmen und ob sich Hinweise auf eine lokale Antikörperproduktion im immunprivilegierten Auge finden lassen. Mittels eines etablierten Protein-Microarray-Verfahrens wurden die Immunreaktionen gegen 40 verschiedene Antigene, wie z.B. Hitzeschock-Proteine oder neuronale Strukturproteine, untersucht. Die Ergebnisse zeigten, dass die detektierten Autoantikörper-Reaktionen gegen mehr als 80% der untersuchten Antigene in beiden Körperflüssigkeiten miteinander übereinstimmen. Verdeutlicht wird hierdurch, dass die Antikörper-basierenden immunologischen Vorgänge im Auge bzw. Kammerwasser, trotz dessen Abschottung vom Blutkreislauf durch die Blut-Retina-Schranke, denen des Serums stark ähneln. Nur vereinzelt lassen sich Hinweise auf eine lokale Antikörperproduktion im Auge finden, wodurch die Bedeutung der detektierten Serumantikörper-Reaktionen für die Glaukomerkrankung belegt wird. rnEin weiterer Schwerpunkt der Arbeit lag auf der Detektion möglicher veränderter Proteinexpressionen in den Retinae und Serumproben von Glaukompatienten, die potentiell zu den neurodegenerativen Prozessen der Glaukompathogenese beitragen. Um die Analyse spezifischer Proteinexpressionen zu ermöglichen, wurde das Verfahren des Antikörper-Microarrays etabliert und auf die Fragestellung angewendet. Untersucht wurden hierbei vor allem die Abundanzen von Komplementproteinen, Zytokinen und Hitzeschock-Proteinen, aber auch die von verschiedenen neuronalen Strukturproteinen. Als Probenmaterial dienten Serum- und Retinaproben von Glaukompatienten, die vergleichend denen von gesunden Probanden gegenübergestellt wurden. Die Analyse erbrachte die Erkenntnis, dass neben der verstärkten Expression von Komplementproteinen in der Retina (z.B. C3, C6) auch im Serum der Glaukompatienten eine erhöhte Konzentration dieser Proteine vorliegt, die im Rahmen der Glaukomerkrankung möglicherweise ebenfalls eine Rolle spielen. Ähnliches konnte für verschiedene Zytokine, wie z.B. TNF-α, IFN-γ oder IL1-β beobachtet werden, die in den untersuchten Retinae von Glaukomprobanden, teilweise auch in den Serumproben der Patienten, in verstärktem Maße detektiert werden konnten. Die erhöhte Produktion von Zytokinen in der Retina ist wahrscheinlich auf die Aktivierung von Gliazellen zurückzuführen, ein Ereignis für das in dieser Arbeit zahlreiche Hinweise gefunden werden konnten. Die Gliaaktivierung wird vermutlich durch apoptotische Prozesse in der Retina ausgelöst, eventuell aber auch durch eine erfolgte Komplementaktivierung. Darüber hinaus konnten mittels eines massenspektrometrischen Verfahrens weitere Expressionsunterschiede verschiedener retinaler Proteine bei Glaukompatienten festgestellt werden. Diese Veränderungen, wie z.B. geminderte Mengen von ROS-eliminierenden Proteinen, wie der Superoxid Dismutase und Peroxiredoxin-2, begünstigen bzw. verstärken sehr wahrscheinlich die neurodegenerativen Prozesse in der Retina von GlaukompatientenrnInwieweit die untersuchten Faktoren kausativ an den neurodegenerativen Prozessen beteiligt sind, bleibt ungeklärt, jedoch untermauert deren Vielzahl die Notwendigkeit, die Ursache der Glaukomerkrankung als komplexe Interaktion und Wechselwirkung verschiedener Komponenten zu betrachten und nicht als einen einzelnen fehlgesteuerten Mechanismus.rn

Identifizierung und Charakterisierung von Protein-Biomarkern beim Glaukom

Das Glaukom ist, nach dem Katarakt, die zweithäufigste Ursache für Erblindungen weltweit mit Milionen von Betroffenen, die von dieser zunächst weitgehend symptomfreien neurodegenerativen Erkrankung heimgesucht werden. Die Möglichkeiten auf dem Feld der Diagnose beschränken sich bislang weitestgehend auf die Messung des Augeninnendrucks und der Beurteilung des Augenhintergrundes durch einen erfahrenen Augenarzt. Eine labordiagnostische Prophylaxe ist bis heute nicht verfügbar, die Zahl unerkannter Erkrankungen dementsprechend hoch. Hierdurch geht wertvolle Zeit verloren, die man für eine effektive Therapie nutzen könnte.rnBezüglich der Pathogenese des Glaukoms geht man heute von mehreren, miteinander wechselwirkenden Pathomechanismen aus, zu denen neben mechanischen Einflüssen durch einen erhöhten IOD auch Hypoxie, verminderte Neutrophinversorgung, Exzitotoxizität, oxidativer Stress und eine Beteiligung autoimmuner Prozesse gezählt werden. Unabhängig vom Pathomechanismus folgt stets die Etablierung umfangreicher degenerativer Prozesse im Sehnervenkopf, den retinalen Ganglienzellen und den Axonen des Sehnerven, die letztlich im irreversiblen Untergang dieser Neuronen münden. Diese pathologischen Prozesse im ZNS hinterlassen auf Proteomebene Spuren, die mithilfe moderner massenspektrometrischer Methoden in Kombination mit multivariaten statistischen Methoden detektierbar und als sogenannte Biomarker-Kandidaten mit definiertem Molekulargewicht darstellbar sind. In dieser Arbeit wurde ein „Workflow“ entwickelt, der es ermöglicht, diese Biomarker-Kandidaten im Blutserum und in der Tränenflüssigkeit in einfachen, reproduzierbaren Schritten zu identifizieren und zu charakterisieren. Abweichend von der etablierten Methotik der Bottom-Up-Proteomics musste hierfür eine Methode entsprechend einer Top-Down-Philosophie entwickelt werden, die es erlaubt, die Spuren des Glaukoms im Proteom zu detektieren und zu charakterisieren.rnDies erfolgte in dieser Arbeit durch sowohl massenspektroskopischen Methoden wie SELDI-TOF® und MALDI-Tof-Tof als auch durch Bead-, Gel- und Flüssigkeits-chromatographisch-basierte Separations und Fraktionierungstechniken.rnDie erfolgreiche Kombination dieser Methoden führte zu Identifikationen einer ganzen Reihe von Biomarker-Kandidaten. Unter den identifizierten Proteinen, die bezüglich ihres korrespondierenden SELDI-Peaks im Massenbereich von Biomarker-Kandidaten liegen, finden sich Zytokine und Effektormoleküle der angeborernen Immunität, stressinduzierbare Kinasen, Faktoren, die zum Schutz der Telomeren dienen, Proliferationsmarker, neuronale Antigene und Transportproteine. Darüber hinaus wurden Komponenten identifiziert, die an der neuronalen Neutrophinversorgung beteiligt sind, neuronale Rezeptoren und Antigene, Komponenten des Komplementsystems und des MHC-I-Komplexes. All diese identifizierten Proteine sind bezüglich ihrer Funktion und möglichen Rolle innerhalb der Pathogenese des Glaukoms detailliert beschrieben und charakterisiert. Dies erlaubt einen umfassenden Einblick in alle Pathomechanismen, denen nach heutigem Kenntnisstand, eine Rolle an der Pathogenese des Glaukoms unterstellt wird.rn

Fabry's disease: otoneurologic findings in twelve members of one family

Fabry's disease corresponds to an inherited disorder transmitted by an X-linked recessive gene. It generates a dysfunction of glycosphingolipid metabolism due to an enzymatic deficiency of alpha-galactosidase activity, resulting in glycosphingolipid deposits in all areas of the body. The clinical (heart, kidney, and central nervous system) manifestations are more severe in hemizygous boys than in heterozygous girls. They appear during childhood or adolescence: acroparesthesia, joint pain, angiokeratoma, corneal dystrophy, hypohydrosis or anhydrosis, and renal failure. The otoneurologic symptoms consist of hearing fluctuation, progressive unilateral or bilateral hearing loss, and episodes of vertigo or dizziness. Otoneurologic findings in 12 of 26 members of the same family are presented: the mother and 9 of her 12 children, as well as 2 of her 14 grandchildren: 4 healthy persons, 4 heterozygous female carriers, and 4 hemizygous male patients. Three of the male patients had fluctuation of hearing, sudden hearing loss, and episodes of vertigo and dizziness. The otoneurologic examinations showed a bilateral cochleovestibular deficit (n = 1), a right cochleovestibular deficit (n = 1), and a bilateral hearing loss combined with a right vestibular deficit (n = 1). Histopathologic evidence of glycosphingolipid accumulation in vascular endothelial and ganglion cells, as well as atrophy of the stria and spiral ligament, might explain the otoneurologic symptoms and findings.

Imaging of total colonic Hirschsprung disease

BACKGROUND: Hirschsprung disease (HD) is a functional obstruction of the bowel caused by the absence of intrinsic enteric ganglion cells. The diagnosis of total colonic HD (TCHD) based on contrast enemas is difficult in newborns because radiological findings vary. OBJECTIVE: To evaluate the radiographic and contrast enema findings in patients with pathologically proven TCHD. MATERIALS AND METHODS: From 1966 to 2007, 17 records from a total of 31 patients with TCHD were retrospectively evaluated for diameter and shape of the colon, diameter of the small bowel, bowel wall contour, ileal reflux, abdominal calcifications, pneumoperitoneum, filling defects, transitional zones and rectosigmoid index. RESULTS: Three colonic patterns of TCHD were found: microcolon, question-mark-shape colon and normal caliber colon. Additional findings included spasmodic colon, ileal reflux, delayed evacuation and abdominal calcifications. Colonic transitional zones were found in eight patients with TCHD. CONCLUSION: The diagnosis of TCHD is difficult to establish by contrast enema studies. The length of the aganglionic small bowel and the age of the patient can influence the radiological findings in TCHD. The transitional zone and the rectosigmoid index can be false-positive in TCHD. The colon can appear normal. Consider TCHD if the contrast enema study is normal but the patient remains symptomatic and other causes of distal bowel obstruction have been excluded.

Expression of adenosine A2a receptor gene in rat dorsal root and autonomic ganglia

The adenosine A2a receptors (A2aR) play an important role in the purinergic mediated neuromodulation. The presence of A2aR in the brain is well established. In contrast, little is known about their expression in the periphery. The purpose of this study was to investigate the expression of A2aR gene in the autonomic (otic, sphenopalatine, ciliary, cervical superior ganglia and carotid body) and in the dorsal root ganglia of normal rat. Hybridization histochemistry with S35-labelled radioactive oligonucleotide probes was used. An expression of A2aR gene was found in the large neuronal cells of the rat dorsal root ganglia. The satellite cells showed no expression of A2aR gene. In the superior cervical ganglion, isolated ganglion cells expressed A2aR. In the carotid body clusters of cells with a strong A2aR gene expression were found. In contrast, the ciliary and otic ganglia did not expressed A2aR gene, and only few small sized A2aR expressing cells were demonstrated in the sphenopalatine ganglion. The discrete distribution of A2aR gene expression in the peripheral nervous system speaks for a role of this receptor in the purinergic modulation of sensory information as well as in the sympathetic nervous system.

High-sensitivity rod photoreceptor input to the blue-yellow color opponent pathway in macaque retina.

Small bistratified cells (SBCs) in the primate retina carry a major blue-yellow opponent signal to the brain. We found that SBCs also carry signals from rod photoreceptors, with the same sign as S cone input. SBCs exhibited robust responses under low scotopic conditions. Physiological and anatomical experiments indicated that this rod input arose from the AII amacrine cell-mediated rod pathway. Rod and cone signals were both present in SBCs at mesopic light levels. These findings have three implications. First, more retinal circuits may multiplex rod and cone signals than were previously thought to, efficiently exploiting the limited number of optic nerve fibers. Second, signals from AII amacrine cells may diverge to most or all of the approximately 20 retinal ganglion cell types in the peripheral primate retina. Third, rod input to SBCs may be the substrate for behavioral biases toward perception of blue at mesopic light levels.

Neural reprogramming in retinal degeneration.

PURPOSE: Early visual defects in degenerative diseases such as retinitis pigmentosa (RP) may arise from phased remodeling of the neural retina. The authors sought to explore the functional expression of ionotropic (iGluR) and group 3, type 6 metabotropic (mGluR6) glutamate receptors in late-stage photoreceptor degeneration. METHODS: Excitation mapping with organic cations and computational molecular phenotyping were used to determine whether retinal neurons displayed functional glutamate receptor signaling in rodent models of retinal degeneration and a sample of human RP. RESULTS: After photoreceptor loss in rodent models of RP, bipolar cells lose mGluR6 and iGluR glutamate-activated currents, whereas amacrine and ganglion cells retain iGluR-mediated responsivity. Paradoxically, amacrine and ganglion cells show spontaneous iGluR signals in vivo even though bipolar cells lack glutamate-coupled depolarization mechanisms. Cone survival can rescue iGluR expression by OFF bipolar cells. In a case of human RP with cone sparing, iGluR signaling appeared intact, but the number of bipolar cells expressing functional iGluRs was double that of normal retina. CONCLUSIONS: RP triggers permanent loss of bipolar cell glutamate receptor expression, though spontaneous iGluR-mediated signaling by amacrine and ganglion cells implies that such truncated bipolar cells still release glutamate in response to some nonglutamatergic depolarization. Focal cone-sparing can preserve iGluR display by nearby bipolar cells, which may facilitate late RP photoreceptor transplantation attempts. An instance of human RP provides evidence that rod bipolar cell dendrite switching likely triggers new gene expression patterns and may impair cone pathway function.