Activated satellite cells in medial rectus muscles of patients with strabismus.

PURPOSE: The goal of this study was to determine whether the medial rectus muscles of patients with a history of medial rectus underaction or overaction show alterations in the process of satellite cell activation when compared with normal age-matched control muscles. METHODS: Medial rectus muscles were obtained with consent from adult patients undergoing surgical resection due to medial rectus underaction or overaction and were prepared for histologic examination by fixation and paraffin embedding. Control muscles were obtained from cornea donor eyes of adults who had no history of strabismus or neuromuscular disease. Cross sections were obtained and stained immunohistochemically for the presence of activated satellite cells, as identified by MyoD immunoreactivity, and the presence of the total satellite cell population, as identified by Pax7 immunoreactivity. The percentages of MyoD- and Pax7-positive satellite cells per 100 myofibers in cross section were calculated. RESULTS: As predicted from results in the literature, MyoD-positive satellite cells, indicative of activation, were present in both the control and resected muscles. In the underacting medial rectus muscles, the percentages of MyoD- and Pax7-positive satellite cells, based on the number of myofibers, were approximately twofold higher than the percentages in the control muscles. In the overacting medial rectus muscles, the percentage of MyoD-positive satellite cells was twofold less than in the control muscles, whereas the percentage of Pax7-positive satellite cells significantly increased compared with that in the control specimens. CONCLUSIONS: The presence of an increased number of activated satellite cells in the resected underacting medial rectus muscles and the decreased numbers of activated satellite cells in the overacting muscles was unexpected. The upregulation in the number of MyoD-positive satellite cells in underacting muscles suggests that there is potential for successful upregulation of size in these muscles, as the cellular machinery for muscle repair and regeneration, the satellite cells, is retained and active in patients with medial rectus underaction. The decreased number of activated satellite cells in overacting MR muscle suggests that factors as yet unknown in these overacting muscles are able to affect the number of satellite cells and/or their responsiveness compared with normal age-matched control muscles. These hypotheses are currently being tested.

Oculomotor Status, Binocular Vision, and Stereoacuity in a Series of Keratoconus Subjects.

Purpose: A group of keratoconus subjects (KG) and a control group (CG) were evaluated for sensory and motor status. We tried to clarify the factors (best-corrected visual acuity [BCVA]), heterophorias, fusional amplitude, anisometropia, astigmatism) that may be associated with a binocular disturbance. Methods: BCVA (logMAR) was measured. Binocular vision was checked using cover tests, striate Maddox, and a 6Δ base-down prism (simultaneous perception), a prism bar (fusion and fusional convergence break point), and Titmus Fly Test (stereopsis). Results: Fifty-four subjects of the KG, 27 men (median 16 years), and 29 of the CG, 15 men (median 20 years), were evaluated. In the KG, 8 (15%) subjects had strabismus. Those whose BCVA in the worse eye was logMAR ≥0.7 had a significantly higher frequency of strabismus and absence of simultaneous perception. Spherical equivalent anisometropia ≥ 1.0 diopter (D) was significantly different in both groups as was the frequency of gross stereopsis. In comparing fine and gross stereopsis in both the KG and the CG, there was a significant difference in the frontal astigmatism between eyes in the KG (P = 0.03) and CG (P = 0.01). Conclusions: In our study, the KG presented a higher frequency of strabismus and impaired binocular vision. Frontal astigmatism was different between groups with gross and fine stereopsis, in both the CG and KG. Future studies are needed to elucidate or reinforce the factors associated with the loss of binocularity in keratoconus. Testing for stereopsis may be helpful to consider in the treatment guidelines for keratoconus.

Increased frequency of activated satellite cells in overacting inferior oblique muscles from humans.

PURPOSE: Strabismus is an oculomotor disorder in which there is a misalignment of the visual axes of the eyes. Inferior oblique muscle (IOM) overaction is a common finding in comitant horizontal strabismus, but its origin is unclear. Recent studies have demonstrated that myogenic satellite cells (SCs) are still activated in adult extraocular muscles, with continuous myonuclear addition in normal uninjured muscles. The objective of this study was to determine whether there are differences in the processes of activation and proliferation of SCs in IOMs of patients with strabismus and IOM overaction and in patients with no history of strabismus. METHODS: Cross sections of IOMs from strabismic and control groups were analyzed immunohistochemically for the presence of MyoD1 and myogenin, specific markers of activated SCs, and for c-Met, which is expressed in quiescent, activated, and proliferating SCs. RESULTS: In overacting IOMs of 26 patients in the strabismic group and 10 patients in the control group, 28.8% and 3.0% of the myofibers, respectively, were associated with MyoD1-positive SC. The frequency of myogenin-positive SC was 30.8% in the strabismic group and 3.6% in the control group, and the frequency of presumptive SCs immunostained for c-Met was 33.6% in the strabismic group and 34.1% in the control group. CONCLUSIONS: The presence of an increased number of activated SCs in overacting IOMs of the strabismic group in contrast to the frequency in the control group resembles the findings detected in developing, regenerating, or hypertrophic muscle tissue. High levels of MyoD1- and myogenin-positive SC in overacting IOMs support the hypothesis that these cells may be involved in alterations in IOM structure correlated with the overaction observed clinically.

[Autologous grafting of extraocular muscles: experimental study in rabbits]. / Transplante autólogo de musculatura ocular extrínseca: estudo experimental em coelhos.

PURPOSE: To evaluate the feasibility of autologous extraocular muscle grafting as a type of muscle expansion surgery. METHODS: The left superior rectus muscle of twenty-nine rabbits was resected and this fragment was attached to the endpoint of the respective right superior rectus (test group). Thereafter, the superior rectus of the left eye was reattached to the sclera (control group). Both groups were examined during different postoperative periods in order to assess their outcomes. RESULTS: The presence of hyperemia was slightly more frequent in the grafted group. Secretion and muscle atrophy were negligible in both groups. Fibrosis was greater in grafted animals. These muscles were weaker than the control muscles, although the force required to split muscular parts was always greater than the physiological one. CONCLUSIONS: This surgical technique was reliable and useful if one intends to achieve muscle expansion without the intrinsic risks of dealing with heterologous/artificial materials.

2-Methyl-1,3-dioxaazaspiro[4.5]decanes as novel muscarinic cholinergic agonists.

Many nonquaternary ammonium muscarinic agonists have been developed over the last few years, but most of the existing compounds (e.g., arecoline, RS-86, AF-30) behave as weak partial agonists at cholinergic receptors in tissues of limited receptor reserve. The current paper describes the synthesis and biochemical assessment of analogues of AF-30 designed to have sufficient conformational freedom to allow greater receptor flexibility and hence activation. The new compounds and important standards were tested in a new biochemical assay designed to measure both receptor affinity and intrinsic activity of each compound and for their ability to stimulate phosphatidylinositol turnover in rat cerebral cortex. Two azaspirodecanes (5a and 5b) were shown to have far greater predicted efficacy than AF-30.

Novel quinuclidine-based ligands for the muscarinic cholinergic receptor.

Recent clinical studies on Alzheimer's patients have implied that only agents displaying high efficacy at the cortical muscarinic receptor have yielded encouraging results. This paper describes the design, synthesis, and biochemical characterization of novel quinuclidine-based muscarinic agonists which can readily penetrate into the central nervous system and which are capable of displaying high efficacy at cortical sites. With use of a biochemical assay capable of measuring receptor affinity and predicting cortical efficacy, it has been discovered that an oxadiazole ring and related heterocycles can function as bioisosteric replacements for the ester moiety found in several known muscarinic ligands. Within this series there exist compounds which span the efficacy range from high-efficacy agonist through partial agonists to antagonists with affinity comparable or superior to that of classical quaternary ammonium ligands. Consistent with recent molecular biology studies, structure-activity trends are interpreted in terms of separate binding sites for agonists and antagonists with H-bonding interactions characterizing agonist behavior and lipophilic binding characterizing antagonist behavior. Thus the aminooxadiazole moiety has structural features which are optimized for an agonist profile.

Relationship between inhibition of cyclic AMP production in Chinese hamster ovary cells expressing the rat D2(444) receptor and antagonist/agonist binding ratios.

1. Radioligand binding assays using [3H]-(-)-sulpiride, in the presence of 1 mM ethylenediaminetetraacetic acid (EDTA) and 100 microM guanylylimidodiphosphate (GppNHp) and [3H]-N0437 were developed to label the low and high agonist affinity states of the rD2(444) receptor (long form of the rat D2 receptor) respectively. The ratios of the affinities of compounds in these two assays (Kapp [3H]-(-)-supiride/Kapp [3H]-N-0437) were then calculated. 2. The prediction that the binding ratio reflected the functional efficacy of a compound was supported by measurement of the ability of a number of compounds acting at dopamine receptors to inhibit rD2(444)-mediated inhibition of cyclic AMP production. When the rank order of the ratios of a number of these compounds was compared to their ability to inhibit the production of cyclic AMP, a significant correlation was seen (Spearman rank correlation coefficient = 0.943, P = 0.01). 3. In conclusion, the sulpiride/N-0437 binding ratio reliably predicted the efficacy of compounds acting at dopamine receptors to inhibit cyclic AMP production mediated by the rD2(444) receptor.

Relative affinities of drugs acting at cholinoceptors in displacing agonist and antagonist radioligands: the NMS/Oxo-M ratio as an index of efficacy at cortical muscarinic receptors.

1. Radioligand binding assays using [3H]-N-methylscopolamine (NMS) and [3H]-oxotremorine M (Oxo-M) have been devised to predict the efficacy of test compounds at muscarinic receptors in rat cerebral cortex. 2. Muscarinic antagonists, including non-selective and both M1- and M2-selective compounds, displayed similar affinity for both binding assays. 3. Full agonists such as carbachol and muscarine possessed a ratio of potencies against the antagonist versus the agonist ligand (NMS/Oxo-M ratio) of greater than 4000. 4. Compounds which have been shown previously to display partial agonist activity in functional assays e.g. pilocarpine and RS86 had intermediate NMS/Oxo-M ratios of 100-150. A second group of compounds which included oxotremorine had somewhat higher ratios (500-1400). 5. The ratio of affinity constants for the two assays predicted the ability of agonists to stimulate cortical phosphatidyl-inositol turnover. 6. These results suggest that the NMS/Oxo-M ratio may be a useful prediction of efficacy for novel compounds acting at cortical muscarinic receptors.

L-689,660, a novel cholinomimetic with functional selectivity for M1 and M3 muscarinic receptors.

1. L-689,660, 1-azabicyclo[2.2.2]octane, 3-(6-chloropyrazinyl)maleate, a novel cholinomimetic, demonstrated high affinity binding (pKD (apparent) 7.42) at rat cerebral cortex muscarinic receptors. L-689,660 had a low ratio (34) of pKD (apparent) values for the displacement of binding of the antagonist ([3H]-N-methylscopolamine ([3H]-NMS) compared with the displacement of the agonist [3H]-oxotremorine-M ([3H]-Oxo-M), in rat cerebral cortex. Low NMS/Oxo-M ratios have been shown previously to be a characteristic of compounds that are low efficacy partial agonists with respect to stimulation of phosphatidyl inositol turnover in the cerebral cortex. 2. L-689,660 showed no muscarinic receptor subtype selectivity in radioligand binding assays but showed functional selectivity in pharmacological assays. At M1 muscarinic receptors in the rat superior cervical ganglion, L-689,660 was a potent (pEC50 7.3 +/- 0.2) full agonist in comparison with (+/-)-muscarine. At M3 receptors in the guinea-pig ileum myenteric plexus-longitudinal muscle or in trachea, L-689,660 was again a potent agonist (pEC50 7.5 +/- 0.2 and 7.7 +/- 0.3 respectively) but had a lower maximum response than carbachol. In contrast L-689,660 was an antagonist at M2 receptors in guinea-pig atria (pA2 7.2 (95% confidence limits 7, 7.4)) and at muscarinic autoreceptors in rat hippocampal slices. 3. The putative M1-selective muscarinic agonist, AF102B (cis-2-methylspiro-(1,3-oxathiolane 5,3')-quinuclidine hydrochloride) was found to have a profile similar to L-689,660 but had up to 100 times less affinity in binding and functional assays.RS-86 (2-ethyl-8-methyl-2,8-diazospiro[4,5]decan 1,3-dionehydrochloride) also had lower affinity than L-689,660, and had no binding selectivity for muscarinic receptor subtypes. RS-86 had a higher NMS/Oxo-M ratio than L-689,660 and was a full agonist at MI,M2 and M3 receptors in the functional pharmacological assays.4. The functional selectivity of L-689,660 in muscarinic pharmacological assays is consistent with the effects of a low efficacy partial agonist in tissues with different effective receptor reserves.

A novel series of non-quaternary oxadiazoles acting as full agonists at muscarinic receptors.

1 A novel series of non-quaternary oxadiazole-based muscarinic agonists demonstrated high affinity for muscarinic receptors. 2. These agonists possessed high efficacy in the nanomolar range at muscarinic receptors in the superior cervical ganglion, atrium and ileum but did not show selectivity across the tissue preparations. 3. Two amino oxadiazoles, one from a quinuclidine series (L-660,863) and one from a 1-azanorbornane series (L-670,207) possessed a high ratio of potency for displacing the binding of [3H]-N-methyl-scopolamine ([3H]-NMS) to potency for displacing the agonist [3H]-oxotremorine-M cortex. 4. The two azanorbornane derivatives L-670,548 and L-670,207 stimulated the turnover of phosphatidylinositol in the cortex with a potency higher than that obtained with any other known muscarinic agonist (ED50 0.26 and 0.18 microM respectively). 5. The maximum response obtained with L-670,207 was greater than that observed for carbachol but was comparable to that of the natural ligand acetylcholine. 6. These oxadiazole muscarinic agonists are among the most potent and efficacious non-quaternary muscarinic agonists ever described.

Direct measurement of muscarinic agents in the central nervous system of mice using ex vivo binding.

Muscarinic agents produce a range of side effects including hypothermia and tremor. Although these responses can be used to estimate the in vivo activity of these muscarinic agents in the central nervous system (CNS), the approach is limited by compensatory feedback mechanisms and the difficulty of equating degree of receptor occupancy to effect. We have developed an ex vivo assay to measure the potency and penetration of muscarinic agents into the CNS. The muscarinic antagonists scopolamine and N-methylscopolamine dose dependently inhibited the ex vivo binding of [3H]oxotremorine-M to homogenates of mouse whole brain membranes. Following intraperitoneal administration these compounds had ED50 values of 2.6 and 26 mg/kg respectively, which were comparable to the doses which inhibited RS86 induced hypothermia in mice. Three muscarinic agonists RS86, pilocarpine and arecoline also demonstrated CNS activity in this assay with ED50 values of 11, 23 and 220 mg/kg. RS86 and pilocarpine additionally showed good penetration into the CNS with estimated values of 1.5 and 0.31% of the administered dose. These values were comparable with the ability of these compounds to induce a centrally mediated hypothermic response. These studies demonstrate a simple, quick and reliable biochemical means of assessing a muscarinic agent's potency and penetration within the CNS.

Muscarinic M1, M2 receptor binding. Relationship with functional efficacy.

A comparison has been made between [3H]pirenzepine binding to the M1 receptor population of rat cerebral cortex and [3H]N-methylscopolamine binding to M2 receptors in rat cardiac membranes. Several standard muscarinic antagonists including trihexyphenidyl HCl, benztropine, biperidin and 4-DAMP (4-diphenylacetoxy-N-methyl piperidine methiodide) showed some selectivity for the M1 binding assay. Dicyclomine and hexahydrosiladifenidol were the only antagonists with a selectivity approaching that of pirenzepine. Gallamine and AFDX-116 were the only M2 (cardiac) selective antagonists. Muscarinic agonists displayed profiles which could be classified into two groups, apparently related to their intrinsic activity. One group displayed apparent selectivity for the heart, with low Hill coefficients and contained full agonists such as acetylcholine. The second group displayed less selectivity, intermediate Hill coefficients and contained partial agonists such as pilocarpine. Thus muscarinic agents can distinguish between different tissues not only on the basis of receptor selectivity, but also by recognition of high and low agonist affinity states. Thus the intrinsic activity of a muscarinic agonist may reflect an apparent but not true receptor-mediated selectivity.

In vivo characterisation of novel efficacious muscarinic receptor agonists.

Although a number of muscarinic agonists have been used in clinical trials for Alzheimer's Disease, many of these compounds are low in potency and have only limited intrinsic efficacy. The present study describes four non-quaternary oxadiazole based muscarinic agonists from a quinuclidine and a 1-azanorbornane series. These displayed up to 1000 fold higher affinity than arecoline and were efficacious muscarinic agonists at cortical receptors. All four compounds produced peripherally mediated salivation and centrally mediated hypothermia at doses 50-50,000 fold lower than arecoline. The most potent was L-670,548, the methyl oxadiazole in the 1-azanorbornane series, which had an ED50 of 0.0016 mg/kg on the hypothermia model. This derivative was also the most potent compound in ex vivo binding studies (ED50 0.0069 mg/kg) and showed excellent brain penetration (3.8% of the administered dose). These derivatives are the first non quaternary efficacious agonists which show good penetration into the CNS (central nervous system), and will prove useful tools in understanding the role of muscarinic receptors in CNS function.

L-687,306: a functionally selective and potent muscarinic M1 receptor agonist.

The oxadiazole L-687,306 is a high affinity muscarinic agonist with a N-methylscopolamine/oxotremorine-M binding profile predictive of a partial agonist. L-687,306 showed marked selectivity in functional pharmacological assays. L-687,306 was a partial agonist at muscarinic M1 receptors in the rat ganglion but a high affinity competitive antagonist at guinea-pig cardiac M2 and ileal M3 muscarinic receptors. This compound gives an opportunity to study receptor reserve involved in muscarinic receptors in vitro and in vivo.

A major problem in strabismus and its possible solution.

PURPOSE: One of the challenges in strabismus is to guarantee stability of the surgical corrections. Re-surgeries are often required even after careful diagnosis, planning, and execution. Several factors contribute to this undesired outcome and the existing management strategies are ineffective. The present alternative is to compensate for their consequences. Ocular rotations are evoked by muscular contractions and relaxations (active forces). During eye movement, periocular tissues are stretched, storing part of the kinetic energy, which may be posteriorly recovered (passive forces), whereas the remaining part of the energy is lost via friction and inelastic deformations (dissipative forces). A method for measuring the forces that cause post-surgery eye drift has not been reported. However, this may be indirectly determined as a function of the respective mechanical variables. The estimated ratio between the kinetic energies of a post-surgery eye drift and a normal pursuit eye movement is ~10(-15). Theoretically, it can be expected that the addition of continuously acting forces of such magnitudes to the oculomotor system might prevent the undesired post-surgery eye movement. METHODS: Several methods for increasing the restraining, dissipative forces to ocular rotations may be conceived. One method is to increase the friction to ocular movements, as for instance, by periocular injection of viscous substances. Another possibility is to use the forces of a magnetic field, which may stabilize the eye in a desired position without avoiding the rotations caused by greater muscular forces acting on it. It has been demonstrated that these forces neutralize the nystagmic movements, whose intensities of mechanical variables are much higher than those of a post-surgery eye drift. Some models of application of this technique are then discussed. Small magnets fixed to the orbit and metallic ferromagnetic elements fixed to the sclera to cover a suitable extension appears to be the best method for providing starting and sliding friction to the oculomotor system. RESULTS: Weak magnetic fields do not cause ocular ill effects. Additionally, the magnetic field may be confined to the elements of the circuit and may not leak. However, the magnetic materials may interfere with magnetic resonance image (MRI) examinations.

A new unity for angular measurements in strabismus.

The practical advantages of quantifying an angle by a ratio of linear lengths instead of arcs of circles has led to the definition of the prism-diopter, a conventional unity for numbering prisms and measuring strabismic deviations. However, a major inconvenience of using prism-diopter unities to express angular measurements is the non-linearity of the scale, which reaches an infinite value for the angle of 90º, then becomes negative, with decreasing magnitudes for increasing angles between 90º and 180º. As a consequence, arithmetical operations and comparisons of angles measured by such unities present errors of very great magnitudes. In order to retain the advantages of defining an angle by straight line dimensions but to diminish the severe inconveniences of this method, a new definition of the prism-diopter is proposed. Here, instead of defining the prism-diopter by the asymmetrical condition, the conception of this new unity is based on a geometrically symmetrical condition; that of the relationship of an isosceles triangle (where the leg is perpendicular to the bisector of the angle and the bisector itself ). The condition of symmetry for the definition of the new unity represents a conceptual advance because it reproduces the already well accepted, conventional criteria for quantifying the value of a prism, that of its minimum deviation. Furthermore, it corresponds to the most commonly observed clinical conditions of binocular balance. The absolute differences between the unitary values of the prism-diopter and that of the new unity are negligible (0.0025%), but the scale of values expressed by the new unity is closer to the ideal scale of angular measurements. (With the new unity, the infinite value is only reached for an angle of 180º and the errors due to arithmetical operations are much smaller.) Numerical examples showing the advantages of using the new unity of angular measurements instead of the prism-diopter are presented. A mathematical generalization of the modifying concept (partition of an angle) with which the new unity is based is also provided.