Inter- and intralaboratory variation of in vitro diffusion cell measurements: an international multicenter study using quasi-standardized methods and materials.

In vitro measurements of skin absorption are an increasingly important aspect of regulatory studies, product support claims, and formulation screening. However, such measurements are significantly affected by skin variability. The purpose of this study was to determine inter- and intralaboratory variation in diffusion cell measurements caused by factors other than skin. This was attained through the use of an artificial (silicone rubber) rate-limiting membrane and the provision of materials including a standard penetrant, methyl paraben (MP), and a minimally prescriptive protocol to each of the 18 participating laboratories. "Standardized" calculations of MP flux were determined from the data submitted by each laboratory by applying a predefined mathematical model. This was deemed necessary to eliminate any interlaboratory variation caused by different methods of flux calculations. Average fluxes of MP calculated and reported by each laboratory (60 +/- 27 microg cm(-2) h(-1), n = 25, range 27-101) were in agreement with the standardized calculations of MP flux (60 +/- 21 microg cm(-2) h(-1), range 19-120). The coefficient of variation between laboratories was approximately 35% and was manifest as a fourfold difference between the lowest and highest average flux values and a sixfold difference between the lowest and highest individual flux values. Intralaboratory variation was lower, averaging 10% for five individuals using the same equipment within a single laboratory. Further studies should be performed to clarify the exact components responsible for nonskin-related variability in diffusion cell measurements. It is clear that further developments of in vitro methodologies for measuring skin absorption are required.

Pronociceptive effects of spinal dynorphin promote cannabinoid-induced pain and antinociceptive tolerance.

Recent studies indicate that sustained opioid administration produces increased expression of spinal dynorphin, which promotes enhanced sensitivity to non-noxious and noxious stimuli. Such increased "pain" may manifest behaviorally as a decrease in spinal antinociceptive potency. Here, the possibility of similar mechanisms in the antinociception of spinal cannabinoids was explored. Response thresholds to non-noxious mechanical and noxious thermal stimuli were assessed. Antinociception was determined using the 52 degrees C tail-flick test. Mice received repeated WIN 55,212-2, its inactive enantiomer, WIN 55,212-3 or vehicle (i.th., bid, 5 days). WIN 55,212-2, but not WIN 55,212-3 or vehicle, produced a time-related increased sensitivity to non-noxious and noxious stimuli. WIN 55,212-2, but not WIN 55,212-3 or vehicle, elicited a significant increase in lumbar spinal dynorphin content at treatment day 5. Increased sensitivity to mechanical and thermal stimuli produced by WIN 55,212-2 was reversed to baseline levels by i.th. MK-801 or dynorphin antiserum; control serum had no effect. WIN 55,212-2, but not WIN 55,212-3 or vehicle, produced dose-related antinociception and repeated administration resulted in antinociceptive tolerance. While MK-801 and dynorphin antiserum did not alter acute antinociception produced by WIN 55,212-2, these substances significantly blocked antinociceptive tolerance when given immediately prior to WIN 55,212-2 challenge on day 5. Daily MK-801 pretreatments, prior to WIN 55,212-2 injection, also produced a significant block of antinociceptive tolerance. These data suggest that like opioids, repeated spinal administration of a cannabinoid CB1 agonist elicits abnormal pain, which results in increased expression of spinal dynorphin. Manipulations that block cannabinoid-induced pain also block the behavioral manifestation of cannabinoid tolerance.

Inhibition of neuropathic pain by selective ablation of brainstem medullary cells expressing the mu-opioid receptor.

Neurons in the rostroventromedial medulla (RVM) project to spinal loci where the neurons inhibit or facilitate pain transmission. Abnormal activity of facilitatory processes may thus represent a mechanism of chronic pain. This possibility and the phenotype of RVM cells that might underlie experimental neuropathic pain were investigated. Cells expressing mu-opioid receptors were targeted with a single microinjection of saporin conjugated to the mu-opioid agonist dermorphin; unconjugated saporin and dermorphin were used as controls. RVM dermorphin-saporin, but not dermorphin or saporin, significantly decreased cells expressing mu-opioid receptor transcript. RVM dermorphin, saporin, or dermorphin-saporin did not change baseline hindpaw sensitivity to non-noxious or noxious stimuli. Spinal nerve ligation (SNL) injury in rats pretreated with RVM dermorphin-saporin failed to elicit the expected increase in sensitivity to non-noxious mechanical or noxious thermal stimuli applied to the paw. RVM dermorphin or saporin did not alter SNL-induced experimental pain, and no pretreatment affected the responses of sham-operated groups. This protective effect of dermorphin-saporin against SNL-induced pain was blocked by beta-funaltrexamine, a selective mu-opioid receptor antagonist, indicating specific interaction of dermorphin-saporin with the mu-opioid receptor. RVM microinjection of dermorphin-saporin, but not of dermorphin or saporin, in animals previously undergoing SNL showed a time-related reversal of the SNL-induced experimental pain to preinjury baseline levels. Thus, loss of RVM mu receptor-expressing cells both prevents and reverses experimental neuropathic pain. The data support the hypothesis that inappropriate tonic-descending facilitation may underlie some chronic pain states and offer new possibilities for the design of therapeutic strategies.

Dynorphin promotes abnormal pain and spinal opioid antinociceptive tolerance.

The nonopioid actions of spinal dynorphin may promote aspects of abnormal pain after nerve injury. Mechanistic similarities have been suggested between opioid tolerance and neuropathic pain. Here, the hypothesis that spinal dynorphin might mediate effects of sustained spinal opioids was explored. Possible abnormal pain and spinal antinociceptive tolerance were evaluated after intrathecal administration of [D-Ala(2), N-Me-Phe(4), Gly-ol(5)]enkephalin (DAMGO), an opioid mu agonist. Rats infused with DAMGO, but not saline, demonstrated tactile allodynia and thermal hyperalgesia of the hindpaws (during the DAMGO infusion) and a decrease in antinociceptive potency and efficacy of spinal opioids (tolerance), signs also characteristic of nerve injury. Spinal DAMGO elicited an increase in lumbar dynorphin content and a decrease in the mu receptor immunoreactivity in the spinal dorsal horn, signs also seen in the postnerve-injury state. Intrathecal administration of dynorphin A(1-17) antiserum blocked tactile allodynia and reversed thermal hyperalgesia to above baseline levels (i.e., antinociception). Spinal dynorphin antiserum, but not control serum, also reestablished the antinociceptive potency and efficacy of spinal morphine. Neither dynorphin antiserum nor control serum administration altered baseline non-noxious or noxious thresholds or affected the intrathecal morphine antinociceptive response in saline-infused rats. These data suggest that spinal dynorphin promotes abnormal pain and acts to reduce the antinociceptive efficacy of spinal opioids (i.e., tolerance). The data also identify a possible mechanism for previously unexplained clinical observations and offer a novel approach for the development of strategies that could improve the long-term use of opioids for pain.

Amlodipine, a calcium channel inhibitor, and cocaine and ethanol's reinforcing effects.

The effects of amlodipine (from 0.1 to 3.0 mg/kg) on rats' pressing for rewarding brain stimulation, with and without cocaine administration, were assessed. None of the doses reliably modified the effects of cocaine. Also, amlodipine was given to two groups of rats taking alcohol: one group that was regularly taking a sweetened alcoholic beverage and the other taking an unsweetened alcoholic beverage. The only discernible effects of amlodipine on alcohol intake were associated with the highest dose and only with rats taking the sweetened beverage. The effects of this high dose could easily be attributable to behavioral toxicity elicited by the dose. In contrast, and confirming previous work, isradipine, another calcium channel inhibitor, produced reliable reductions on both cocaine's and alcohol's reinforcing effects. Despite the similarity of isradipine and amlodipine, isradipine apparently has some unique features with respect to cocaine and alcohol.

Suicide under the age of eighteen: a 10-year retrospective study.

The number of suicides in the pediatric age group is rising, and death investigators need to be aware of the common scenarios, risk factors, and victims as they investigate such cases to properly assign the cause and manner of death. We reviewed all pediatric cases referred to the Medical University of South Carolina, Forensic Section, from January 1988 through January 1998. Thirty-one cases of pediatric suicide were analyzed with regards to age, gender, race, cause of death, surrounding circumstances, and past history. Sixty-eight percent of victims were aged 16 or 17 years, 84% were male, 68% were white, 78% used firearms to commit suicide, 81% were found within close vicinity of their home, and 26% had a documented history of mental illness. Suicide is a manner of death that is often difficult for the public to accept, especially in pediatric cases. We report our findings in this 10-year retrospective study to better understand this entity and work toward the prevention of future cases.

Papillo-renal syndrome. An inherited association of optic disc dysplasia and renal disease. Report and review of the literature.

A family is described in which the father and son had chronic renal disease of early onset and bilateral optic nerve dysplasia. A further son, known to have microphthalmos died of renal disease in childhood. Optic nerve changes included coloboma in the father and Handmann's optic nerve anomaly, a condition resembling the morning glory syndrome (M.G.S.), in the son. There was electrodiagnostic and visual field evidence of optic nerve dysfunction even where acuity was relatively unaffected. The son developed central serous retinopathy, a condition frequently encountered in association with optic nerve dysplasias, including M.G.S.

Histologic changes in porcine eyes treated with high-intensity focused ultrasound.

High-intensity focused ultrasound (HIFU) has recently been described in the treatment of patients with glaucoma. However, despite previous studies in animal and mathematical models, the mechanisms of action are not completely understood. We therefore undertook a histologic study of 15 porcine eyes to evaluate the effect of HIFU and, in particular, the changes seen after sequential insonification on previously treated areas of sclera. We demonstrated initial scleral swelling that was followed by scleral thinning. The scleral thinning could be maximized by a second superimposed course of HIFU. The most consistent change in the ciliary body was necrosis of the pars plana. Some treated animals developed a hemorrhagic retinal detachment that we believe to be due to the presence of a circumferential blood vessel, which runs in the ora serrata of the pig but which is not present in humans. Based on our observations, possible mechanisms of action of this treatment are discussed.

Ultrasonic tissue characterization and histopathology in tumor xenografts following ultrasonically induced hyperthermia.

Cells derived from human skin malignant melanoma were implanted subcutaneously in athymic nude mice. Tumors which developed at the implant site were treated with ultrasonically induced hyperthermia at 49 degrees C for 30 min. Tumors were scanned with a computerized diagnostic ultrasound system before and after treatment. Light (LM) and electron (EM) micrographs of tumors were obtained after scanning. Changes in ultrasonic tissue characterization parameters following treatment were well correlated with histopathologic changes observed in tumors. The results are significant in terms of clinical application of ultrasonically induced hyperthermia for treatment of intraocular tumors and the noninvasive monitoring of tumors by use of diagnostic ultrasound.

Ultrasonic hyperthermia and radiation in the management of intraocular malignant melanoma.

Hyperthermia and radiation were used in combination to treat four patients with choroidal malignant melanoma. This technique uses ultrasonically induced hyperthermia synergistically with radiation to destroy tumor cells. The lower levels of radiation used should avoid the late vascular and inflammatory complications seen in conventional radiation therapy. Tumors were scanned by a computerized diagnostic ultrasound system before treatment and assigned an acoustic tissue type on the basis of a statistical comparison of their ultrasound backscatter spectrum with spectra of tumors of known pathologic status. During the follow-up period, the longest of which was 15 months, all tumors demonstrated regression patterns consistent with choroidal tumors of the same acoustic tissue types treated with conventional radiation therapy.

Treatment of glaucoma with high-intensity focused ultrasound.

This report is a summary of results for 170 eyes of patients with refractory glaucoma treated with high-intensity focused ultrasound. The results are analyzed in terms of the effectiveness of various treatment regimens, complications, and classifications of the patient population according to such factors as age and etiology. The mean pretreatment intraocular pressure (IOP) for the ensemble of patients treated with optimal intensity levels was 38.6 mmHg. Whereas only 10% of these patients had an IOP of 25 mmHg or less prior to treatment, 90% had an IOP of 25 mmHg or less within 3 months of treatment. At 1 year after a single treatment, 65% of patients still maintained intraocular pressures of 25 mmHg or less, and 56% had pressures of 22 mmHg or less. The effectiveness of retreatment of failed or unresponsive cases was also investigated and found to have a degree of success comparable to that of initial treatments.

Effect of hyperthermia on experimental choroidal melanoma.

Thirty-five rabbit eyes were implanted subchoroidally with Greene's hamster melanoma. When the tumours reached a base diameter of 5 mm, they were treated with ultrasonically induced hyperthermia with a range of temperatures and exposure durations (43-67 degrees C and 75 s to 60 min). Of the 23 treated eyes examined two months after treatment eight showed complete regression of the tumour. Seven showed initial tumour regression, but there was subsequent regrowth of tumour round the margins of the original mass. In eight eyes the tumour continued to grow, though in some cases the rate of growth appeared to be slower than in the controls. In contrast, in all untreated animals the tumour grew to fill the vitreous cavity. These preliminary findings indicate that ultrasonically induced hyperthermia can be an effective local treatment of this intraocular tumour model.

Treatment of experimental lens capsular tears with intense focused ultrasound.

High-intensity focused ultrasound was employed to seal lens capsular tears in a rabbit model. Ultrasound therapy was applied either contiguously, thereby completely covering the tear, or in a discrete exposure pattern around the tear. Both methods prevented the formation of a generalised cataract. This was in contrast to results observed in a group of control (untreated) animals which all developed generalised lens opacities. Each control animal also developed a local lens opacity at the site of the capsular tear, as did half the animals treated with the discrete pattern. No animal treated with contiguous exposures developed any local or generalised traumatic-type cataract other than the small lens opacity immediately produced by the treatment. These treatment cataracts would not constitute a significant impediment to vision so long as they did not fall on the visual axis.

Therapeutic ultrasound in the treatment of glaucoma. II. Clinical applications.

Focused, high-intensity therapeutic ultrasound was used to treat 69 selected patients with uncontrollably elevated intraocular pressure (IOP). This new technique selectively thins scleral collagen, and produces focal damage to the ciliary epithelium. These tissue modifications provide a reduction in IOP pressure to 25 mmHg or less in 83% of patients with a minimum three-month follow-up period.

The penetration of Sorbinil, an aldose reductase inhibitor, into lens, aqueous humour and erythrocytes of patients undergoing cataract extraction.

Two studies to investigate the penetration of the aldose reductase inhibitor, Sorbinil, were conducted. In the first study, 24 diabetic patients undergoing intracapsular extraction were randomised into three groups on a double masked basis. In the week immediately preceding the operation, all patients were requested to take two capsules daily before breakfast. Each capsule contained 200 mg Sorbinil, 100 mg Sorbinil, or a placebo. On measuring Sorbinil levels in lens, plasma and erythrocytes using HPLC, three clearly defined groups of patients were obtained. In one group no Sorbinil was detected, in the second group there were moderate levels of Sorbinil, while the third group had significantly higher levels of Sorbinil. The ratio of erythrocyte/plasma Sorbinil was 0.225, while the ratio for lens/plasma was 0.7, for both groups where Sorbinil was detected. In a second study, 20 patients were treated topically with a single dose of 0.5 mg ophthalmic Sorbinil at times ranging from 0-14 hr preoperatively. Sorbinil was detected in both lens and aqueous. Transport into the lens was complete within about 2 hr, and although aqueous levels were negligible after 6 hr, Sorbinil persisted up to 14 hr in the lens. Three out of 16 patients taking Sorbinil orally developed a maculopapular rash with pyrexia approximately 8 days after commencing the drug. No side effects were noted in any patients given the topical ophthalmic preparation.

Inherited recurrent corneal erosion.

The causes of inherited recurrent corneal erosion may be classified into two major groups: (1) Those associated with primary epithelial dystrophies, including the Franceschetti type, a newly described epithelial rosette dystrophy, basement membrane disorder, and Meesmann's dystrophy, (2) Those associated with the anterior limiting membrane, stromal, and endothelial dystrophies. There is also a third group, including epidermolysis bullosa, in which recurrent erosion attacks are part of a wider systemic disorder. Attention is drawn to the age at onset, presentation, morphological features, and progression of each disorder.

Frequency of faecal Klebsiella aerogenes in patients with ankylosing spondylitis and controls with respect to individual features of the disease.

A study of 59 patients with definite ankylosing spondylitis and 41 comparable hospital outpatients with fractures has been undertaken to determine if the presence of faecal Klebsiella aerogenes is related to clinical activity of the spinal disease and its extraspinal features. The frequencies of fecal K. aerogenes were similar in both patients and controls and were not significantly related to spinal disease activity. Careful inquiry about antibiotic treatment, dietary habits, and hospitalisation did not significantly influence the results. A significant association was found between the presence of faecal K. aerogenes and both acute non-granulomatous anterior uveitis (P less than 0.01) and peripheral synovitis in HLA B27 positive patients (P less than 0.05). These results suggest that K. aerogenes may have an aetiological role in the development of non-granulomatous anterior uveitis and peripheral arthritis in patients with ankylosing spondylitis but do not lend support to this organism having such a role in the spinal disease itself.