Tolerance and cross-tolerance to morphine-like stimulus effects of mu opioids in rats.

The purpose of these experiments was to examine the relationship of agonist relative efficacy to the pattern of tolerance and cross-tolerance to the morphine-like stimulus effects of three opioid agonists. Rats were trained to discriminate 3.2 mg/kg morphine from saline under fixed-ratio 15 schedule of food reinforcement. Morphine, nalbuphine, and fentanyl produced dose-dependent increases in morphine-like stimulus effects and decreases in response rates. Repeated treatment with 20 mg/kg per day morphine increased the ED50 for stimulus control by fentanyl, morphine, or nalbuphine two-, four-, or 40-fold, respectively. Repeated treatment with 64 mg/kg per day nalbuphine increased the ED50 for stimulus control for morphine by two-fold, but lower or higher treatment doses had no significant effect. Treatment with 100 mg/kg per day nalbuphine increased the ED50 for nalbuphine by six-fold. Repeated treatment with 0.22 mg/kg per day fentanyl increased the ED50 for stimulus control by fentanyl or morphine by approximately two-fold. Comparisons among treatment conditions suggested that magnitude of tolerance to morphine-like stimulus effects did not vary as an inverse function of the relative efficacy of the agonist used for repeated treatment. Rather repeated morphine and fentanyl treatments produced comparable tolerance, whereas repeated nalbuphine treatment did not evoke substantial tolerance. Comparisons within treatment conditions, however, suggested that magnitude of tolerance may vary inversely with relative efficacy of the agonist tested for morphine-like stimulus effects. During treatment with morphine or fentanyl, greater tolerance was observed to the morphine-like stimulus effects of the lower efficacy agonist relative to the higher efficacy agonist.

In vivo apparent pA2 analysis in rats treated with either clocinnamox or morphine.

Experiments tested the hypothesis that loss of agonist potency or effectiveness following irreversible antagonist or chronic agonist treatment may result from affinity changes at mu opioid receptors. Apparent affinity of naltrexone or nalbuphine for mu opioid receptors was measured in vivo in rats treated with either a single dose of the irreversible antagonist clocinnamox or repeated doses of morphine. Apparent affinity of each antagonist was estimated from its potency as an antagonist of discriminative stimulus or rate-decreasing effects of morphine in rats trained to discriminate 3.2 mg/kg morphine and saline. In control rats, apparent pA2 values for naltrexone and nalbuphine were 7.5-7.6 and 5.3, respectively. In clocinnamox-treated rats, apparent pA2 values for naltrexone were 7.2-7.7, suggesting that clocinnamox treatment did not alter affinity of naltrexone for sites through which morphine exerts behavioral effects. In rats treated repeatedly with morphine, apparent pA2 values for nalbuphine were 5.1-5.3, suggesting that repeated morphine treatment did not alter affinity of nalbuphine for these sites. The observation that neither clocinnamox nor repeated morphine treatment altered in vivo affinity estimates for naltrexone or nalbuphine, respectively, suggests that the reductions in agonist potency produced by these treatments do not result from changes in affinity at mu opioid receptors.

Humoral response to oligomeric human immunodeficiency virus type 1 envelope protein.

The humoral immune response to human immunodeficiency virus type 1 (HIV-1) is often studied by using monomeric or denatured envelope proteins (Env). However, native HIV-1 Env complexes that maintain quaternary structure elicit immune responses that are qualitatively distinct from those seen with monomeric or denatured Env. To more accurately assess the levels and types of antibodies elicited by HIV-1 infection, we developed an antigen capture enzyme-linked immunosorbent assay using a soluble, oligomeric form of HIV-1IIIB Env (gp140) that contains gp120 and the gp41 ectodomain. The gp140, captured by various monoclonal antibodies (MAbs), retained its native oligomeric structure: it bound CD4 and was recognized by MAbs to conformational epitopes in gp120 and gp41, including oligomer-specific epitopes in gp41. We compared the reactivities of clade B and clade E serum samples to captured Env preparations and found that while both reacted equally well with oligomeric gp140, clade B seras reacted more strongly with monomeric gp120 than did clade E samples. However, these differences were minimized when gp120 was captured by a V3 loop MAb, which may lead to increased exposure of the CD4 binding site. We also measured the ability of serum samples to block binding of MAbs to epitopes in gp120 and gp41. Clade B serum samples consistently blocked binding of oligomer-dependent MAbs to gp41 and, to a slightly lesser extent, MAbs to the CD4 binding site in gp120. Clade E serum samples showed equivalent or greater blocking of oligomer-dependent gp41 antibodies and considerably less blocking of CD4-binding-site MAbs. Finally, we found that < 5% of the antibodies in clade B sera bound to epitopes present only in monomeric gp120, 30% bound to epitopes present in both monomeric gp120 and oligomeric gp140, and 70% bound to epitopes present in oligomeric gp140, which includes gp41. Thus, captured oligomeric Env closely reflects the antigenic characteristics of Env protein on the surface of virions and infected cells, retains highly conserved epitopes that are recognized by antibodies raised against different clades, and makes it possible to detect a much greater fraction of total anti-HIV-1 Env activity in sera than does native monomeric gp120.

Glaucoma as a cause of poor vision in severe retinopathy of prematurity.

Despite surgical reattachment of the retina in infants with severe retinopathy of prematurity (ROP), visual acuity may decline. We performed gonioscopy and applanation tonometry on 26 eyes of 17 infants with severe ROP who were randomly selected and followed prospectively for 2 years. Of these 26 eyes, one eye of one patient had only peripheral retinocryopexy for threshold stage III disease and had an attached retina before enrollment into the study. Sixteen infants subsequently underwent vitrectomy, scleral buckle, or both. Retinal reattachment of the posterior pole was present in at least one eye each of 10 of the 17 infants. Glaucoma was diagnosed in 5 of the 10 patients; all 5 showed a decrease in visual acuity of greater than 1 octave. Of the 5 infants without glaucoma, 3 showed visual improvement, and 2 remained the same. Although we studied a small number of cases, our results suggest that glaucoma may be a cause of visual decline in infants with severe ROP. A decline in visual acuity in infants with severe ROP and surgical retinal reattachment may warn us of glaucoma, as this diagnosis is difficult to make in these small infants.

Intraocular pressure determination in infants with severe retinopathy of prematurity.

We determined intraocular pressure (IOP) in 55 premature infants (122 determinations) with stage V retinopathy of prematurity (ROP) by applanation and indentation tonometry corrected for scleral rigidity and found a modest correlation of r = 0.52 between readings. Chronologic age and the scleral rigidity coefficient were the most significant factors affecting the closeness in readings. Our findings suggest that the applanation method of obtaining IOP values is preferable in infants with ROP because this method is less affected by technical error and the variable nature of premature infant sclera. Frequent examination may be necessary to detect an early IOP increase, especially in infants who have had successful retinal reattachment through vitreoretinal techniques.

Anterior segment evaluation of infants with retinopathy of prematurity.

To understand the mechanisms of glaucoma in retinopathy of prematurity (ROP), anterior segment evaluation is essential. The authors prospectively examined the anterior segment of 27 eyes of 17 premature infants with stages IV and V ROP. Twenty-six eyes received no previous surgery or treatment. Schiøtz and applanation tonometry were performed. Structural evaluation of each anterior segment was conducted by biomicroscopy and Koeppe gonioscopy. In the 26 eyes, angle closure of greater than 180 degrees was noted in 3 (12%). The authors noted prominent Schwalbe's line in 4 eyes (15%), high iris convexity in 15 (58%), hypopigmentation of the iris root in 19 (73%), translucent matrix in the angles ("Barkan's-type" membrane) in 18 (69%), posterior synechiae in 16 (62%), visible iris or angle vessels in 12 (46%), and pigment clumping in the angle recess in 12 (46%). This study identified structural abnormalities in the anterior segment of ROP infants, including pathologic changes and anatomic features that could have a developmental origin.

Trabecular meshwork recovery after phagocytic challenge.

The short and long term response of the trabecular meshwork to a phagocytic challenge and the response of the meshwork to different types of foreign particles was studied by injecting one eye of 25 adult cats with a phagocytic agent (zymosan, blood, or latex microspheres) while the fellow eye received a control solution. Eyes were examined histologically at various intervals from one day to five months after infusion. Active trabecular cell phagocytosis and changes in cell shape were found with all agents. The extent of these changes varied with the agent used. Zymosan caused marked changes and inflammation, with numerous macrophages found throughout the meshwork. Trabecular cell migration and cell loss occurred, although it was often difficult to distinguish macrophages from rounded trabecular cells. The meshwork eventually recovered from this inflammatory insult, as trabecular lamellae became less edematous and once again acquired a lining of trabecular cells. Blood and latex microspheres caused less disruption, with microspheres often found in otherwise normal appearing cells. Trabecular cellularity was quantitated after the blood and the zymosan infusions. No cell loss was observed after the blood infusion, while zymosan-infused eyes had an initial 15% cell loss (p less than .04) when compared with fellow control eyes. This zymosan-associated trabecular cell loss may have been due to phagocytosis, inflammation, or a combination of both. The cell loss had recovered by the end of 150 days (p less than .02), as trabecular cell numbers in experimental eyes became comparable to fellow control eyes.

Phagocytosis by trabecular meshwork cells: sequence of events in cats and monkeys.

Trabecular meshwork cells have well-established phagocytic properties. However, the sequential stages of this process have not been studied in detail. Zymosan particles, yeast cell walls 3 micron in diameter, were either injected or perfused into the anterior chambers (AC) of cats and monkeys. Glutaraldehyde (3%) was then infused into the AC at 1-, 6-, 17-, 30- and 60 min and the fate of the particles observed by transmission electron microscopy. After contact with zymosan particles, trabecular meshwork cells demonstrated short cytoplasmic extensions at contact sites and an increased number of cytoplasmic organelles. Internalization of the particles occurred by 6 min in the cat and by 17 min in the monkey. In both species similar events occurred following internalization of zymosan: trabecular cells, laden with particles, rounded up, detached from their underlying collagenous beams and migrated towards Schlemm's canal or the aqueous plexus. By 1 hr the individual trabecular cells completed the entire sequence of phagocytic events although some cells were always observed in earlier stages of the process. These data document for the first time the timing of the cellular events that take place during zymosan particle phagocytosis by trabecular meshwork cells.

Effect of vitamin A on glycoconjugate: synthesis in the trabecular meshwork. A preliminary report.

We studied the effect of vitamin A on glycoconjugate synthesis in trabecular meshwork (TM) cells in vivo. The aqueous flow system of two cats and one monkey was exposed in vivo to retinoic acid dissolved in dimethyl sulfoxide (DMSO). One hour prior to enucleation, [3H]glucosamine was injected into all eyes. The tissue was subsequently processed for autoradiography. The density of grains (grains micron-2) in the vitamin A-treated eyes of Cats I and II was 77% and 30% less, respectively, than the density of grains in the control eyes. In the monkey the grain density was 56% less in the experimental eye compared with the control eye. These data suggest that exogenous vitamin A suppresses glycoconjugate synthesis in TM cells in vivo.

Calculations of flow resistance in the juxtacanalicular meshwork.

The structure of the juxtacanalicular meshwork (JCM) was analyzed morphometrically, and the resulting data were used to calculate the resistance to flow through this tissue. Two models of the JCM were presented and compared. In the first (Model A), aqueous humor was assumed to flow via open channels within a solid framework, while, in the second (Model B), these open spaces were assumed to be filled with extracellular matrix gel. An expression giving the resistance of such a gel as a function of gel concentration was presented and tested on corneal and scleral stroma. Morphometry of normal and glaucomatous human eyes showed that Model A underpredicted the resistance of the JCM by factors of 10-100, suggesting that a GAG or proteoglycan gel may control the flow resistance of this tissue. This was supported by Model B, which showed that measured bulk concentrations of GAGs were consistent with gel concentrations needed to account for the estimated resistance of the JCM in vivo. Some limitations and implications of Model B were discussed.

Response of exfoliation glaucoma to laser trabeculoplasty.

The comparative response of exfoliation (EF) glaucoma and primary open-angle glaucoma (POAG) to laser trabeculoplasty (LTP) was studied retrospectively. The effectiveness of argon laser trabeculoplasty (LTP) was compared in 26 eyes of patients with EF glaucoma (uncontrollable by maximally tolerated medical therapy) and 28 eyes of patients with POAG (controls). 64% of the POAG group and 46% of the EF glaucoma group received treatment of the entire angle in two divided sessions in order to reduce the intraocular pressure (IOP) to less than or equal to 22 mmHg. All second treatments were performed at least four to six weeks after the initial session. Evaluation of the postoperative course of IOP by means of the Kaplan-Meier curve for data analysis indicated that EF patients 'failed' at a faster rate than POAG patients after both the initial and consecutive laser treatments. The rate of failure in the EF group, however, was greater following the initial 180 degrees treatment. Although patients with EF glaucoma have a large initial reduction in intraocular pressure after LTP, to increase the probability of maintaining intraocular pressures of less than or equal to 22 mmHg consecutive 180 degrees treatments should be considered.

Establishment of calf trabecular meshwork cell cultures.

Trabecular meshwork cell cultures have been grown from excised calf aqueous outflow pathway tissue using two different methods. In one method, tissue explants were placed directly in culture dishes and primary cultures were established by those cells migrating from the tissue. In the second method, a cell suspension was generated by incubating trabecular tissue strips with 0.1% pronase, and the washed cells were used to seed primary cultures. The cell cultures generated by each method appeared to progress through morphological and biochemical changes characteristic of the cell type, although at different respective rates. Comparisons of the cell morphologies, biosynthetic activities, and growth characteristics of the respective subcultures suggested that similar cell populations were obtained by each method. These studies further suggested that sufficient quantities of calf trabecular cells could be grown in culture to permit biochemical and pharmacological studies of trabecular-cell metabolism.

Factors influencing glycosaminoglycan synthesis by calf trabecular meshwork cell cultures.

When cell or explant cultures were established from excised calf aqueous outflow pathway tissue and subsequently labeled with radioactive precursors of glycosaminoglycans [( 3H]glucosamine and [35S]sulfate), hyaluronic acid was the predominant glycosaminoglycan produced initially. As the cultures reached confluence, sulfated glycosaminoglycans became more prominent products. Further investigation revealed that the cultured trabecular cells could synthesize all of the glycosaminoglycans found in isolated calf trabecular tissue once a critical cell density had been attained. Increasing concentrations of fetal bovine serum in the culture medium stimulated glycosaminoglycan accumulation without altering the quantitative distribution of the various glycosaminoglycan products. Treatment of confluent monolayers with enzymes capable of degrading extracellular matrix molecules caused marked changes in the distribution of glycosaminoglycans produced by calf trabecular-cell cultures. The results suggest that calf trabecular cell cultures can be used to study the dynamics of extracellular matrix production and turnover under controlled experimental conditions.

Pigmentary dispersion syndrome and pigmentary glaucoma. A prospective study of the natural history.

The natural evolution of pigmentary dispersion syndrome and pigmentary glaucoma was studied in 55 patients for six to 43 months (mean, 27 months). Active dispersion of pigment was observed in 45 eyes in 31 patients and was associated with worsening of glaucoma in 32 eyes. Dispersion of pigment was defined as active when there was increase in iris transillumination, increase in corneal pigmentation, or appearance of pigment granules on the surface of the lens in the pupil. There were no differences in the frequency of active dispersion of pigment and worsening of glaucoma comparing patients aged less than 44, 45 to 64, and 65 or more years. This study demonstrates that active dispersion of pigment is clinically detectable, is correlated with elevation of intraocular pressure, and continues to occur in older patients.

Shock-wave effect on anterior segment structures following experimental neodymium:YAG laser iridectomy.

The short-pulse laser may prove superior to the argon laser in producing noninvasive iridectomies. Little is known of the effects of the laser shock-wave on anterior segment structures adjacent to the iris. To investigate this question, iridectomies were produced in two owl monkeys using a neodymium (Nd):YAG laser and tissues were evaluated by light microscopy and scanning and transmission electron microscopy. For purposes of comparison, one eye received an iridectomy produced by an argon laser. Results indicate that while clean iris colobomas can be produced with a well-focused Nd:YAG laser, its shock-wave affects tissues in both the trabecular meshwork and corneal endothelium if the iridectomy is located within 0.8 mm of the limbus. No damage to the trabecular meshwork or corneal endothelium was evident after the argon laser iridectomy.

Race as a risk factor for progressive glaucomatous damage.

Retrospective analysis of black and white men with glaucoma indicated that open-angle glaucoma may be a more severe disease in blacks than in whites. Disk damage and visual field loss at initial diagnosis appeared to be worse in the eyes of blacks. Progression of disease as evidenced by further disk damage and visual field loss was more marked in the eyes of blacks. Furthermore, at initial diagnosis blacks were younger than whites, suggesting an earlier onset of open-angle glaucoma among blacks.

A morphologic and morphometric analysis of the aqueous outflow system of the developing cat eye.

The cellular and tissue changes accompanying the development and growth of the aqueous outflow system of the cat were investigated by quantitative light microscopy and by scanning and transmission electron microscopy. As in primates, the trabecular beams and sheets of the cat aqueous outflow system developed by reorganization of cells and extracellular matrix within the tissue filling the anterior chamber angle recess. Enlargement and coalescence of intercellular spaces gave rise to intertrabecular channels. From 3 to 9 days after birth, communications were established between the anterior chamber and intertrabecular spaces by perforation and resorption of tissue which initially covered the angle apex and appeared to be a peripheral extension of Descement's membrane and the corneal endothelium. Macrophage-like cells could be involved in this process. A rapid increase in the volume of the intertrabecular spaces and in the number of trabecular cells coincided with the opening of the trabecular meshwork to the anterior chamber. The trabecular meshwork grew 150-fold in volume from birth to adulthood, mainly as a result of a similar-fold expansion of its connective tissue components. The volume of the intertrabecular spaces increased 24-fold and trabecular cell number increased 14-fold during this same period. The disproportionate increase in volume of the various components of the trabecular meshwork was responsible for the decreased cell density and rarefaction displayed by this tissue as development progressed. Development of the aqueous outflow system of the cat is thus a complex, but highly co-ordinated, process, that depends on continued proliferation of cells and extracellular matrix, a progressive ordering of these components, and selective atrophy and removal of specific tissue components.

Distribution of glycosaminoglycans in the aqueous outflow system of the cat.

Although glycosaminoglycans (GAGs) have been postulated to play a role in the regulation of intraocular pressure, structural localization of specific varieties of GAGs in the outflow system is necessary before their precise role can be determined. In this study, the outflow system of the cat was stained with ruthenium red to identify GAGs with the electron microscope. The composition of the ruthenium red-stainable material was determined by predigestion of tissues with testicular hyaluronidase, neuraminidase, or papain. Testicular hyaluronidase-sensitive GAGs were located on the surfaces of the endothelial cells in the trabecular meshwork and aqueous plexus, within their basal laminae, and in the amorphous tissue of the trabecular beams and tissue adjacent to the aqueous plexus. Collagen and elastic fibers throughout the outflow system were also associated with ruthenium red-stainable material that was resistant to testicular hyaluronidase. Connective tissue GAGs, but not endothelial cell-associated GAGs, were demonstrated to be complexed to protein, since they were disrupted by papain treatment. Neuraminidase-sensitive material (sialoglycoprotein) was identified only on the lumenal surface of the endothelial cells of the aqueous plexus. The complex distribution of GAGs and order polyanions in the outflow system of the cat suggests that the these macromolecules may serve more than one function.

Exfoliation glaucoma: a quantitative perfusion and ultrastructural study.

The aqueous outflow systems of both eyes from a patient with well documented exfoliation syndrome were studied postmortem by quantitative perfusion and scanning and transmission electron microscopy. This provided a rare opportunity to correlate clinical and postmortem findings. the right eye was phakic with early glaucoma. The left eye was aphakic with moderately severe glaucoma prior to the cataract surgery. The eyes were fixed at a perfusion pressure of 25 mm Hg. In both eyes the spaces of the corneoscleral trabecular meshwork were open and mostly free of exfoliation material. The trabecular cells appeared normal and contained no exfoliation material. Failure to take up exfoliation material contrasts with the known tendency of these cells to take up pigment and other particles. The main pathology involved destruction of Schlemm's canal and accumulation of exfoliation material in the juxtacanalicular region of both eyes. The changes in Schlemm's canal and the juxtacanalicular region appeared sufficient to account for increase in resistance to aqueous outflow.