Thomas W. Mittag

Detection of Anti-Acetylcholine Receptor Factors in Serum and Thymus from Patients with Myasthenia Gravis

Since the blood and thymus of patients with myasthenia gravis may contain inhibitors of neuromuscular transmission that affect acetylcholine receptors of striated muscle, we used denervated rat muscle to test for inhibitors in 43 serums and 18 thymus glands from such patients. Seven per cent of serums inhibited the binding of 125l alpha-bungarotoxin to triton-solubilized receptors; 65 per cent interfered with binding of toxin-labeled receptors to concanavalin-A coupled to Sepharose gel, and 85 per cent formed IgG-receptor complexes detectable by immunoprecipitation. Serum inhibitory activity varied widely among patients with similar clinical manifestations and was not correlated with duration of myasthenia gravis or thymectomy. Among thymus extracts, 44 per cent were inhibitory in the concanavalin-A binding assay, whereas 72 per cent contained anti-receptor IgG. Thus, serums from patients with myasthenia gravis contain more than one anti-receptor factor.

Maintaining Mitochondrial Membrane Impermeability: An Opportunity for New Therapy in Glaucoma?

Apoptosis may contribute to retinal ganglion cell loss in glaucoma and glaucoma models. Recent research has suggested that mitochondrially dependent apoptosis signaling may contribute to apoptosis in a rat model of glaucoma involving chronic increases in intraocular pressure. In some forms of apoptosis, mitochondrially dependent signaling involves increases in mitochondrial membrane permeability and the mitochondrial release of factors that signal for cell degradation. Opening of a multi-protein, mitochondrial megapore is one factor that contributes to the increased permeability and some anti-apoptotic proteins, particularly BCL-2 and BCL-X(L), bind at the megapore and facilitate megapore closure and reduce increases in mitochondrial membrane permeability. Phosphorylated protein kinase B (Akt) serves as an integrator for cellular survival signals and facilitates the megapore actions of BCL-2 and BCL-X(L), which could protect retinal ganglion cells against insults that induce apoptosis. Several anti-apoptotic agents are being evaluated for use in glaucoma, including brimonidine and propargylamines, which oppose mitochondrially dependent apoptosis through pathways involving phosphorylated Akt.

Reduced ocular pulse amplitude in low tension glaucoma is independent of vasospasm

Purpose: A vascular basis for the pathogenesis of primary open angle glaucoma has been postulated for many years. Defects in the regulation of ocular blood flow may be the initiating factor in this group of multifactorial diseases. This study was designed to evaluate the effect of vasospasm on ocular pulse amplitude (OPA) in low tension glaucoma (LTG) patients.Methods: OPA, using the Langham Ocular Blood Flow (OBF) System, applanation intraocular pressure (IOP), systemic blood pressure and heart rate were measured and vasospasm was determined by a fingernail capillary blood flow test.Results: OPA (mmHg) in the LTG patients with a vasospastic reaction (LTG-V, 1.4 ± 0.1, n= 17) was not significantly (p>0.09) different when compared with non-vasospastic LTG patients (LTG-NV, 1.5 ± 0.2, n= 15) but was significantly (p<0.001) reduced in LTG-V and LTG-NV patients when compared with matched healthy control subgroups (2.3 ± 0.2 and 2.4 ± 0.3, respectively). IOP (mmHg) in LTG-V (13.3 ± 0.4) and LTG-NV (13.2 ± 0.5) patient groups was not significantly (p>0.05) different when compared with each other, but was significantly (p<0.05) lower when compared with matched control subgroups (15.0 ± 0.3 and 15.2 ± 0.4, respectively). Haemodynamic parameters were not significantly different from controls.Conclusion: The abnormality in choroidal perfusion indicated by a reduction in OPA in all LTG patients is unrelated to the presence or absence of vasospasm.

Influence of physical exercise and nifedipine on ocular pulse amplitude

Abstract• Background: Ocular pulse amplitude (OPA) was measured to investigate the influence of peripheral vasoconstriction and vasodilatation on choroidal perfusion in healthy volunteers and to determine whether low OPA in low-tension glaucoma (LTG) patients is associated with a vasospastic reaction and its response to the calcium channel blocker nifedipine.• Methods: OPA was determined using the Langham ocular blood flow (OBF) system, applanation intraocular pressure (IOP), systemic blood pressure, and heart rate were measured, and ocular perfusion pressure was calculated before and after exercise and smoking in 12 otherwise nonsmoking, healthy volunteers and prior to and for 3 months after initiation of nifedipine therapy in 32 LTG patients with and without a vasospastic reaction as manifested by a nailfold capillary blood flow test.• Results: Exercise significantly (P<0.05) increased heart rate, systolic blood pressure and ocular perfusion pressure, while it significantly (P<0.05) reduced IOP and diastolic blood pressure. However, OPA was not significantly (P>0.1) affected by changes in these parameters. Smoking significantly (P<0.05) increased systolic and diastolic blood pressure, heart rate, and ocular perfusion pressure but did not significantly (P>0.09) alter OPA. There were two distinct LTG subtypes, with and without a vasospastic reaction. Only those with a vasospastic reaction showed a significant (P<0.001) increase in OPA after 3 months of nifedipine treatment, while all other parameters tested were not significantly altered.• Conclusion: Despite affecting ocular and systemic perfusion parameters, exercise and smoking did not alter OPA, suggesting functional isolation, i.e. autoregulation of the choroidal and/or ophthalmic artery circulation in healthy volunteers. Low OPA in LTG was increased by nifedipine only in vasospastic LTG patients, suggesting different, vasotonus-related pathologies. Calcium channel blockers and other vasodilators may be useful in vasoreactive LTG patients with reduced OPA.

Nitric oxide synthase activity in tissues of the bovine eye

Abstract• Background: Nitric oxide synthase (NOS) is present in many ocular tissues where it may have different physiological functions. This warrants a thorough characterization of NOS activity in the eye. • Methods: NOS distribution and its biochemical properties were determined in the retina, choroid, ciliary processes (CP), and trabecular meshwork (TM). • Results: Retinal NOS required NADPH (diphenyleneiodonium, a flavoprotein inhibitor, which inhibited enzyme activity with an IC50 of 0.36 μM, FAD (40 μM), FMN (40 μM), and BH4 (4 μM) as cofactors for optimal activity. Ocular NOS appeared to be regulated by free divalent cations, since its activity was inhibited by EDTA (slopes >3.0 and IC50 values of 12.8, 19.7, and 53 μM, respectively). Ocular NOS required calmodulin, since NOS activity was inhibited by trifluoperazine (calmodulin inhibitor, IC50=41 μM). NOS activity is widely distributed in the eye, (choroid >reinta >CP >TM) and is mainly cytosolic (70–95%).l-Arginine analogs inhibited NOS in the retina, choroid, and TM. In all three tissues,NG-methyl-l,-arginine displayed the highest affinity for inhibition (IC50=0.2–0.7 μM) followed by canavanine (IC50=13–33 μM), while aminoguanidine only weakly inhibited NOS (IC50=93–179 μM). • Conclusion: In all tissues, the order of potency of inhibition points to the presence of constitutive rather than inducible NOS. Moreover, it is possible that TM contains more than a single form of NOS.

Alpha-adrenergic antagonists: Correlation of the effect on intraocular pressure and on α2-adrenergic receptor binding specificity in the rabbit eye

Six alpha-adrenergic antagonists, which have a range of selectivities for alpha 1- and alpha 2-adrenoreceptor subtypes, were compared with respect to their ability to reduce intraocular pressure (IOP) after topical application to the rabbit eye, and their affinity and selectivity for alpha 2-adrenoreceptors, as determined by binding to membranes prepared from rabbit iris-ciliary body. A routine assay for alpha 2-adrenoreceptors using [3H]-rauwolscine was developed for this purpose. ICB contained 200-300 fmol (mg protein)-1 alpha 2-adrenoreceptors which represents approximately two-thirds of the total number of alpha-adrenoreceptor sites present in this tissue. All six antagonists bound at alpha 2-adrenergic receptor sites in an apparently simple competitive manner. The Kd for three of the drugs was about 10 nM (rauwolscine, yohimbine, WB-4101) and the Kd for the other three was greater than 3500 nM (prazosin, corynanthine, thymoxamine). However, all six antagonists were effective ocular hypotensive agents when given topically in a 50 microliter dose of 1% (w/v) concentration. The ability of alpha-adrenergic antagonists to lower IOP in the rabbit did not correlate with a single alpha-receptor subtype and appears to involve at least two separate mechanisms, one mediated by alpha 2-adrenergic receptors and one mediated by alpha 1-adrenergic receptors.

Okuläre Pulsamplitude bei okulärer Hypertension und verschiedenen Glaukomformen

The present study was designed to investigate choroidal perfusion in primary open-angle glaucoma patients with statistically elevated (HPG) and statistically normal (NPG) intraocular pressure (IOP) and in ocular hypertensive volunteers (OHT) before and after application of topical antiglaucomatous drugs. When compared to age-matched healthy controls, ocular pulse amplitude (OPA) and IOP were significantly increased in OHT and unchanged in HPG; in NPG, the IOP was unchanged, and the OPA was significantly reduced. The topical antiglaucomatous drugs used in this study reduced IOP, but did not increase OPA. When compared to HPG and NPG, OHT subjects showed increased choroidal perfusion which may help to prevent glaucomatous optic nerve damage.

Gene Expression Changes in Areas of Focal Loss of Retinal Ganglion Cells in the Retina of DBA/2J Mice

Purpose. To determine whether differences in gene expression occur between areas of focal retinal ganglion cell (RGC) loss and of relative RGC preservation in the DBA/2 mouse retina and whether they can provide insight into the pathophysiology of glaucoma. Methods. Areas of focal RGC loss (judged by lack of Fluorogold labeling; Fluorochrome, Denver, CO), adjacent areas with relative RGC preservation in DBA/2 retina, and Fluorogold-labeled retina from DBA/2(-pe) (pearl) mice were dissected and used for microarray analysis. RT-PCR and immunoblot analysis were used to confirm differential gene expression. Bioinformatic analysis was used to identify gene networks affected in the glaucomatous retina. Results. Microarray analysis identified 372 and 115 gene chip IDs as up- and downregulated, respectively, by 0.5-fold in areas of RGC loss. Differentially expressed genes included those coding for cytoskeletal proteins, enzymes, transport proteins, extracellular matrix (ECM) proteins, and immune response proteins. Several genes were confirmed by RT-PCR. For at least two genes, differential protein expression was verified. Bioinformatics analysis identified multiple affected functional gene networks. Pearl mice appeared to have significantly different gene expression, even when compared with relatively preserved areas of the DBA/2 retina. Conclusions. Regional gene expression changes occur in areas of focal RGC loss in the DBA/2 retina. The genes involved code for proteins with diverse cellular functions. Further investigation is needed to determine the cellular localization of the expression of these genes during the development of spontaneous glaucoma in the DBA/2 mouse and to determine whether some of these gene expression changes are causative or protective of RGC loss.

The effects of topically applied epinephrine and timolol on intraocular pressure and aqueous humor cyclic-AMP in the rabbit☆

Abstract Intraocular pressure and aqueous humor cyclic-AMP concentrations were measured in albino rabbits following topical treatment of one eye with a single dose of 2% epinephrine alone, 0·5% timolol alone, or epinephrine after timolol pretreatment. Most animals demonstrated a significant hypotensive response 6 hr after epinephrine treatment, which lasted at least an additional 6 hr. However, aqueous humor c-AMP was significantly elevated 30 min after epinephrine treatment, peaked between 60 and 240 min, and declined to baseline by 6 hr before a significant ocular hypotensive response was noted. Timolol treatment alone had no significant effect on either intraocular pressure or aqueous c-AMP. However, in epinephrine-treated animals which were pre-treated with timolol, the c-AMP response was blocked, with no significant alteration of the hypotensive response. Previous reports in the literature suggested a causal relationship between elevated aqueous humor c-AMP and fall in IOP. On the basis of the present work, this relationship is questionable.

Noninvasive determination of intraocular pressure (IOP) in nonsedated mice of 5 different inbred strains.

BackgroundNoninvasive intraocular pressure (IOP) measurement in mice is critically important for understanding the pathophysiology of glaucoma. Rebound tonometry is one of the methods that can be used for obtaining such measurements. We evaluated the ability of the rebound tonometer (RT) to determine IOP differences among various mouse strains and whether differences in corneal thickness may affect IOP measurements in these animals. Materials and MethodsFive different commonly used mouse strains (BALB/C, CBA/CAHN, AKR/J, CBA/J, and 129P3/J) were used. IOP was measured in eyes from 12 nonsedated animals (6 male and 6 female) from each strain at 2 to 3 months of age using the RT. IOPs were measured in all animals, on 2 different days between 10 AM and 12 PM. Subsequently, a number of eyes from each strain were cannulated to provide a calibration curve specific for that strain. Tonometer readings for all strains were converted to apparent IOP values using the calibration data obtained from the calibration curve of the respective strain. For comparison purposes, IOP values were also obtained using the C57BL/6 calibration data previously reported. IOP for the 5 strains, male and female animals, and the different occasion of measurement were compared using repeat measures analysis of variance. The central corneal thickness (CCT) of another group of 8 male animals from each of the 5 strains was also measured using an optical low coherence reflectometry (OLCR) pachymeter modified for use with mice. CCT values were correlated to mean IOPs of male animals and to the slopes and intercept of individual strain calibration curves. ResultsNoninvasive IOP measurements confirm that the BALB/C strain has lower and the CBA/CAHN has higher relative IOPs than other mouse strains while the AKR/J, the CBA/J, and the 129P3/J strains have intermediate IOPs. There is a very good correlation of apparent IOP values obtained by RT with previously reported true IOPs obtained by cannulation. There was a small but statistically significant difference in IOP between male and female animals in 2 strains (129P3/J and AKR/J) with female mice having higher relative IOPs. No correlation between CCT and IOP was detected. CCT did not correlate with any of the constants describing the calibration curves in the various strains. ConclusionsNoninvasive IOP measurement in mice using the RT can be used to help elucidate IOP phenotype, after prior calibration of the tonometer. CCT has no effect on mouse IOP measurements using the RT.