Exercise-induced hyperemia unmasks regional blood flow deficit in experimental hindlimb ischemia.

UNLABELLED: Many experimental models of hindlimb ischemia are characterized by spontaneous and rapid normalization of resting muscle blood flow (BF) rates which complicates the long-term evaluation of angiogenic therapies to reverse limb ischemia. We tested the hypothesis that peroneal nerve stimulation in an ischemic hindlimb would increase the oxygen (O(2)) demand and BF rate, thereby unmasking a severe blood flow deficit that is not apparent at rest. METHODS: Ischemia was induced in adult rats by ligation of the left common iliac, femoral arteries, and their branches. Peroneal nerves were stimulated to allow measurement of exercise-induced regional BF rates with fluorescent microspheres. Hemodynamics were monitored. Fluorescent microspheres were injected before and after 5 min of nerve stimulation 3, 10, and 24 days postischemia. The tibialis anterior (TA) and gastrocnemius (GC) muscles and skin were harvested and weighed, and fluorescence was measured. BF rate was calculated as milliters per minute per gram of tissue and compared to normal muscle and skin of unoperated control rats. In order to determine the accuracy of BF rate measurements in ischemic muscle when <400 microspheres was delivered per specimen, 3 rats were studied by simultaneous injection of 4 x 10(5) blue and 1 x 10(5) yellow-green fluorescent microspheres. The correlation coefficient between the number of different colored microspheres delivered was measured. RESULTS: The ischemia caused atrophy of the TA and GC muscles. The mean muscle mass of the ischemic TA and GC as a percentage of total body weight decreased over time vs control [TA 0.13 +/- 0.05% vs 0.25 +/- 0.03%, P < 0.05; GC 0.51 +/- 0.27% vs 0.70 +/- 0.07%, P = 0.07 at 24 days (24D)]. Despite clinical evidence of severe hindlimb ischemia in experimental groups, i.e., pressure sores, muscle atrophy, and weakness, resting BF rates were not significantly different from those of control. The BF rate of the TA was of 0.11 ml/min/g after 3D of ischemia, 0.14 ml/min/g after 10D, and 0.13 ml/min/g after 24D. The mean BF rate in normal muscle of unoperated controls was 0.16 ml/min/g (P > 0.05). However, the exercise-induced hyperemia in the skeletal muscle was significantly blunted in all of the ischemic groups. The unoperated control TA had a greater than 10-fold increase in BF to 1.95 ml/min/g in response to exercise while the ischemic TA had no increase in BF at 3D, 2-fold increase at 10D, and a 5-fold increase at 24D. Parallel findings were noted in the GC muscles. There was no significant difference in the BF rate in the skin. The accuracy of this microsphere technique in measuring very low BF rates found in ischemic muscle was supported by the significant correlation coefficient (r = 0.99) comparing two quantities of microspheres injected simultaneously. CONCLUSION: Despite clinical signs of severe hindlimb ischemia, resting BF rates in the ischemic groups were not significantly decreased. Peroneal nerve stimulation resulted in up to 10-fold increase in BF rate and unmasked a severe deficit in vascular reserve in the ischemic groups. Resting BF rate is not always an accurate reflection of the flow deficit in models of critical limb ischemia, and this model of exercise-induced hindlimb hyperemia may allow better long-term evaluation of angiogenic therapies designed to reverse critical limb ischemia.

Adenoviral-mediated gene transfer of ICP47 inhibits major histocompatibility complex class I expression on vascular cells in vitro.

PURPOSE: Many viruses have evolved mechanisms to evade detection by the host immune system. The herpes simplex gene ICP47 encodes a protein that binds to the host antigen-processing transporter, inhibiting the formation of major histocompatibility complex class I (MHC-I) antigens in infected cells. MHC-I antigen expression is also important in acute allograft rejection. This study was designed to quantitate the effect of adenoviral-mediated gene transfer of ICP47 on MHC-I cell surface expression of human vascular cells. We hypothesized that the transduction of vascular cells with a replication-incompetent adenoviral vector that was expressing ICP47 (AdICP47) would inhibit constitutive and inducible MHC-I expression and thereby reduce the rate of cytolysis of ICP47-transduced vascular cells by sensitized cytotoxic T lymphocytes (CTL). METHODS: A replication-incompetent adenoviral vector, AdICP47, was created to express ICP47 driven by the cytomegalovirus immediate early promoter. Cultured human vascular endothelial and smooth muscle cells and human dermal fibroblasts were transduced with either AdICP47 or the control empty vector AddlE1. Cell surface constitutive and gamma-interferon-induced MHC-I expression were quantitated by flow cytometry. A standard 4-hour chromium release cytotoxicity assay was used to determine the percent cytolysis of transduced and nontransduced endothelial cells by sensitized CTL. Finally, to quantitate the specificity of the effect of ICP47 on MHC-I expression, adhesion molecule expression was quantitated in both transduced and nontransduced cells. RESULTS: Constitutive MHC-I expression in AdICP47-transduced endothelial cells was inhibited by a mean of 84% +/- 5% (SEM) in five experiments. After 48 hours of exposure to gamma-interferon, AdICP47-transduced cells exhibited a mean of 66% +/- 8% lower MHC-I expression than nontransduced cells. Similar inhibition in MHC-I expression was achieved in AdICP47-transduced vascular smooth muscle cells and dermal fibroblasts. Percent cytolysis of AdICP47-transduced endothelial cells by CTL was reduced by 72%. Finally, the specificity of the effect of transduction of ICP47 on vascular cell MHC-I expression was confirmed by a lack of significant change in either constitutive or tumor necrosis factor-induced vascular cell adhesion molecule/intercellular adhesion molecule expression. CONCLUSION: Transduction of vascular cells with AdICP47 strongly inhibits both constitutive and inducible MHC-I expression in human vascular cells. AdICP47-transduced cells exhibited a substantial reduction in cytolysis by CTL. Thus AdICP47 transduction holds promise as a technique to characterize the role of MHC-I expression in acute vascular allograft rejection in vivo and as a potential therapeutic intervention.

A blinded trial of local recombinant tissue factor pathway inhibitor versus either local or systemic heparin in a vein bypass model.

PURPOSE: Tissue factor pathway inhibitor (TFPI), an endogenous protease, is a potent inhibitor of the extrinsic pathway of coagulation. To determine whether TFPI could be used as an alternative to systemic heparin and dextran in vein bypass grafting procedures, we compared the efficacy of these agents in a blinded trial using a pig model of lower extremity vein bypass grafting. METHODS: Yorkshire pigs (60 to 75 kg) were divided into four groups of five each: systemic heparin (5 ml 10(3) U heparin, 50 ml intravenous dextran, and 10 U heparin/ml flush), local heparin (5 ml saline solution, 50 ml intravenous dextran, and 10 U heparin/ml flush), recombinant TFPI (rTFPI) (5 ml saline solution, 50 ml intravenous saline, and rTFPI 90 micrograms/ml flush), and control (5 ml and 50 ml intravenous saline and intravenous phosphate-buffered saline solution flush). The pigs were anesthetized and the lesser saphenous vein was harvested and reversed to construct a bypass from the common femoral artery to the saphenous artery at the hock. Each pig received two intravenous infusions before cross-clamping, and the artery and vein were flushed locally according to the protocol for each treatment group. Coagulation parameters were drawn 30 minutes after cross-clamping. The surgical team was blinded as to the pigs' treatment group throughout the protocol. RESULTS: The time from initial infusion until bypass completion averaged 80 minutes. Conduit patency rates at 7 days were as follows: four of five in the rTFPI group, three of five in the systemic heparin group, one of five in the local heparin group, and zero of five in the control group. The activated partial thromboplastin time was elevated (50.1 +/- 13.8 seconds) with systemic heparin but not in the other groups. CONCLUSIONS: Local administration of TFPI prevents thrombosis as effectively as systemic heparin and dextran and is superior to local heparin flush plus dextran (p = 0.02). Thus local TFPI offers an excellent alternative to systemic heparin plus dextran and avoids the risks of systemic anticoagulation.

Role of hepatic lipase in the uptake and processing of chylomicron remnants in rat liver.

The participation of hepatic lipase in the initial uptake and processing of chylomicron remnants was studied in the isolated, perfused rat liver. Hepatic lipase activity was either reduced by preperfusion of livers with heparin or inhibited with specific rat hepatic lipase antibodies. [3H]palmitate-labeled chylomicron remnants were recirculated through control and treated livers for 15 min; the livers were then flushed, homogenized, and endosome-rich fractions were isolated. Depletion of hepatic lipase activity by both methods reduced the uptake of chylomicron remnants and hydrolysis of their component triglycerides by perfused rat livers, but at the same time significantly increased the rate of endocytosis of those chylomicron remnants taken up. We conclude that hepatic lipase facilitates, but is not essential for, the initial uptake of chylomicron remnants by rat liver. Furthermore, endocytosis of chylomicron remnants does not require binding to hepatic lipase or the associated hydrolysis of remnant lipids.

Penetrating keratoplasty after ocular trauma.

We reviewed the records of 41 patients who underwent penetrating keratoplasty for a corneal opacity caused by trauma between Jan. 1, 1983, and Dec. 31, 1988. Most of the patients were young males whose average age was 35 years. Common tools accounted for many injuries (14 of 41, 34%). Sixteen injuries (39%) were work-related. Thirty-six patients (88%) had corneal scars from a perforating injury, four patients (10%) had corneal edema, and one patient (2%) required emergency penetrating keratoplasty for extensive tissue loss. Of the 39 patients with one year of follow-up, 31 (82%) maintained clear corneal grafts. Rejection occurred in nine of 39 patients (23%) and only three (33%) of the rejections resolved. Visual outcome was favorable with 20 patients (51%) attaining best-corrected postoperative visual acuity of 20/20 to 20/40, nine patients (23%) attaining visual acuity of 20/50 to 20/100, and ten patients (26%) attaining visual acuity of 20/200 or worse. Preoperative retinal disease (six of 39, 15%), astigmatic errors (five of 39, 13%), graft failures (four of 39, 10%), and postoperative glaucoma (12 of 39, 31%) adversely influenced the outcome. Although the prevalence of postoperative complications is relatively high, good visual results can be obtained in patients who undergo penetrating keratoplasty after ocular trauma.

Indications for and outcomes of repeat penetrating keratoplasty.

We reviewed data on 150 repeat corneal transplants performed between 1983 and 1988 at Wills Eye Hospital. Minimal postoperative follow-up was six months. The most common diagnoses before regraft were pseudophakic bullous keratopathy (29 of 150), aphakic bullous keratopathy (28 of 150), Fuchs' dystrophy (21 of 150), failed graft (17 of 150), and keratoconus (14 of 150). Herpes simplex keratitis, interstitial keratitis, chemical burns, traumatic scars, and corneal dystrophies accounted for most of the remaining regrafts. Of the 150 regrafts, 39 (26%) failed during the study period. Almost half of the regraft failures occurred within six months, and over four fifths occurred within 18 months. Of the 111 eyes with clear grafts, final visual acuity data were available for 68 eyes (minimal follow-up, two years; mean, 3.3 years). A best-corrected visual acuity of 20/20 to 20/40 (22 of 68) or 20/50 to 20/100 (27 of 68) was achieved in most patients.

Diagnosis and treatment of chronic postoperative bacterial endophthalmitis.

Bacterial endophthalmitis has been recognized as a significant cause of chronic, delayed-onset inflammation following extracapsular cataract extraction (ECCE) with posterior chamber intraocular lens implantation. The diagnosis of chronic postoperative bacterial endophthalmitis should be suspected in any case of chronic, refractory inflammation following ECCE. Although successful treatment may require surgical intervention in some cases, others may respond to antibiotic therapy alone.

Clinical indications for and procedures associated with penetrating keratoplasty, 1983-1988.

We reviewed the preoperative clinical indications and associated surgical procedures for 2,299 penetrating keratoplasties performed at our institution from 1983 through 1988. Pseudophakic bullous keratopathy was the most common indication overall, accounting for 526 cases (23%). A marked increase was noted in the incidence of pseudophakic bullous keratopathy as an indication for penetrating keratoplasty beginning in 1985. The association of anterior chamber intraocular lenses in eyes with pseudophakic bullous keratopathy undergoing penetrating keratoplasty increased from 19 of 43 cases (44%) in 1983 to 79 of 108 cases (73%) in 1988. The incidence of intraocular lens exchange at the time of penetrating keratoplasty in cases of pseudophakic bullous keratopathy increased from six of 43 (14%) in 1983 to 63 of 108 (58%) in 1988. Other major indications for penetrating keratoplasty included Fuchs' dystrophy (375 cases, 16%), keratoconus (348 cases, 15%), aphakic bullous keratopathy (331 cases, 14%), and regraft (233 cases, 10%). Cataract extraction, with or without intraocular lens implantation, was combined with penetrating keratoplasty in 397 of 1,532 phakic eyes (26%). The incidence of triple procedure (penetrating keratoplasty, cataract extraction, and intraocular lens implantation) increased from 27 of 248 phakic eyes (11%) in 1983 to 68 of 258 phakic eyes (26%) in 1988.

Membrane binding sites for plasma lipoproteins on endosomes from rat liver.

Highly purified endosomal membranes from rat liver, enriched in receptors for a number of macromolecules taken up into hepatocytes via the coated pit/endosome/lysosome pathway [including the receptor for low density lipoproteins (LDL)], were used to characterize binding sites for lipoproteins containing apolipoprotein E. In endosomal membranes from livers of estradiol-treated rats, in which LDL receptors are induced manyfold, two high-affinity binding sites were found for two apolipoprotein E-rich lipoproteins: very low density beta-lipoproteins (beta-VLDL) from cholesterol-fed rabbits and rat chylomicron remnants. One of these sites, binding to which is inhibited by 30 mM EDTA, appears identical to the LDL receptor by ligand and immunoblotting and other characteristics. The other site, highly resistant to EDTA, does not bind LDL. Binding to the EDTA-resistant site, however, is readily inhibited by heparin (as is the LDL receptor) and also by antisera prepared against rat or bovine LDL receptor. The distribution of the EDTA-resistant site among early endosomes, late endosomes, and endosome-derived receptor-recycling membranes is similar to that of the LDL receptor and other recycling receptors. The LDL receptor was present in endosomal membranes from livers of untreated rats at about 10% of the level found in membranes from estradiol-treated rats, but the EDTA-resistant site was barely detectable. No saturable binding of beta-VLDL that could not be inhibited by antisera to the LDL receptor could be detected in endosomal membranes from livers of either untreated or estradiol-treated rats. The EDTA-resistant site may be a modified form of the LDL receptor that recognizes apolipoprotein E but not the B apolipoprotein of LDL. Alternatively, it may be a distinct receptor sharing immunological determinants with the LDL receptor, specialized for the endocytosis of certain lipoproteins containing apolipoprotein E, including chylomicron remnants.

Pseudophakic bullous keratopathy.

We reviewed the records of all patients with pseudophakic bullous keratopathy (271 eyes, 251 patients) seen during a six-month period to determine predisposing factors, associated problems, current management, and visual outcome. Pseudophakic bullous keratopathy was associated most frequently with anterior chamber intraocular lenses in general (155 of 271), and with Leiske style lenses in particular (100 of 271). It was associated with a visual acuity of 20/200 or less in 206 eyes and a visual acuity of counting fingers or less in 129 of the eyes at the initial examination. Penetrating keratoplasties had been performed in 189 of the eyes. After penetrating keratoplasty, 108 of 189 of the eyes had a visual acuity of 20/200 or less (mean follow-up, 15 months). Visual acuity improved with longer follow-up, and among patients with a minimum follow-up of two years, 23 of 36 eyes had a visual acuity of 20/100 or better. Most grafts were clear (145 of 189). Pseudophakic bullous keratopathy was associated with marked visual loss, which was permanent despite clear grafts in 29 of 92 eyes followed-up for one year or longer.

Diagnosis and treatment of chronic postoperative bacterial endophthalmitis.

Chronic postoperative bacterial endophthalmitis has recently assumed a prominent role in differential diagnosis of inflammation following extracapsular cataract extraction with posterior chamber intraocular lens implantation. The optimal diagnostic and therapeutic approach to this entity has not yet been clearly defined. We present a case of chronic postoperative Propionibacterium acnes endophthalmitis in which the diagnosis was made by anterior chamber paracentesis, and topical, periocular, and systemic antibiotic therapy resolved the inflammation. Anterior chamber paracentesis for aerobic and anaerobic cultures may be an appropriate initial diagnostic step in suspected cases. While successful treatment may require surgical intervention in some cases, others may respond to antibiotic therapy alone.

Isolation and characterization of three endosomal fractions from the liver of estradiol-treated rats.

Three distinct endosomal fractions were isolated in high purity from livers of estradiol-treated rats. Each fraction had characteristic physical and ultrastructural properties, but the lipid composition and major proteins of their membranes were similar and differed from those derived from the Golgi apparatus. Injected radioiodinated low density lipoproteins accumulated first in the fraction of intermediate density and later in the low density fraction. The latter was composed almost exclusively of lipoprotein-filled multivesicular bodies, most of which had a single membranous appendage. The fraction of intermediate density was composed of lipoprotein-filled vesicles that were smaller than multivesicular bodies and also had membranous appendages. The high density fraction was composed of membranes resembling the appendages of the two vesicular fractions. All three fractions were enriched in receptors for low density lipoproteins and asialoglycoproteins, but receptor concentrations were considerably reduced in multivesicular bodies. The fraction of intermediate density may represent the compartment of uncoupling of receptor and ligand (CURL) described by Geuze et al. [Geuze, H. J., Slot, J. W., Strous, G. J. A. M., Lodish, H. F. & Schwartz, A. L. (1983) Cell 32, 277-287]. CURL vesicles may lose some of their appendage as multivesicular bodies are formed. The high density fraction then may represent a receptor-recycling compartment.

The effects of prolonged phacoemulsification time on the corneal endothelium.

The question of whether or not phacoemulsification causes significant corneal endothelial damage has been studied in many ways. In this study, we used videotaped specular microscopy and nitroblue tetrazolium (NBT) staining to assess cell damage under conditions in which the two most commonly blamed sources of damage--probe tip trauma and lens fragments--cannot be implicated. After 15 minutes of irrigation, aspiration, and ultrasound in a modified corneal viewing and storage (CVS) chamber, the four test corneas showed less than 5% cell damage as assessed by NBT staining, which was no more than in the control. In one case, an unexpected air bubble on the endothelium caused a loss of endothelial cells. Because small air bubbles are common during phacoemulsification, and because such air bubbles may represent a cause of endothelial cell loss, the endothelial damage caused by air bubbles during phacoemulsification merits further study.

Melanocytoma: nomenclature, pathogenesis, natural history and treatment.

A melanocytoma (magnocellular nevus) is a benign pigmented tumor that may arise wherever uveal melanocytes are present. The most commonly observed site is on or adjacent to the optic nerve head. In the past this tumor was frequently misdiagnosed as a malignant melanoma and many unnecessary enucleations were carried out. Its clinical features are now well recognized and, fortunately, specimens of melanocytomas are rarely received in ocular pathology laboratories today. A review of the literature concerning this tumor is presented, with special reference to the changing concepts in nomenclature, pathogenesis, natural history, and treatment that have evolved in the past one hundred years. We also define the clinical and histopathological features of this tumor.

Histopathologic correlative study of Kelman-style flexible anterior chamber intraocular lenses.

Three of 16 eyes in which Kelman flexible anterior chamber lenses were implanted were obtained for postmortem examination, and the lenses and globes were analyzed histopathologically. Thirteen additional lenses, removed surgically secondary to various complications, were studied by scanning electron microscopy. All of the lenses ere well finished and none showed evidence of degradation or cracking of the polymethylmethacrylate optics or foot plates. Some of the lenses showed deposition of inflammatory debris or erythrocytes, reflecting a clinical history of uveitis. In some cases, the problems seemed to be related to variations in surgical technique or improper sizing.

Choroiditis in infantile periarteritis nodosa.

The ocular pathologic findings were determined in a case of fatal infantile periarteritis nodosa (IPN). To our knowledge, this case is the first histologically documented report of choroidal inflammation associated with this disease. Since the clinical and pathologic findings of IPN and Kawasaki's disease (KD) are identical, these findings should be sought in cases of KD.

Malignant transformation of an optic nerve melanocytoma.

A 47-year-old woman who had had a flat, hyperpigmented lesion of the optic disc and juxtapapillary choroid for at least 17 years experienced acute visual loss. This was caused by rapid growth of a pigmented tumour emanating from the original lesion. Histopathological examination of the enucleated globe revealed a large malignant melanoma originating from a melanocytoma (magnocellular nevus) within the optic nerve and peripapillary choroid. This case represents a very rare instance of malignant transformation of a normally benign tumour.

Complications of intraocular lenses. A historical and histopathological review.

Recent improvements in intraocular lens (IOL) design, manufacturing techniques, and surgical techniques have greatly reduced the incidence of complications following implantation, and many authors now consider IOL implantation to be among the most safe and effective major surgical procedures. However, adverse reactions are still seen--some as late sequelae of earlier IOL designs and implantation techniques and some as sequelae of more recent implantations using "state-of-the-art" lenses and surgical techniques. Complications may be due to various factors, including surgical technique, IOL design, or the inability of some eyes with preexisting disease to tolerate an implant. The authors trace the evolution of IOLs since Ridley's first implant, summarizing the modifications in lenses and surgical techniques that were made as complications were recognized. They then review the clinical and histopathological features of selected cases from more than 200 IOLs and/or globes removed due to IOL-related complications and studied in the University of Utah Ocular Pathology Laboratory. It is hoped that this review will provide insights into the pathogenesis of IOL complications, enhancing the current success of implant procedures and stimulating further basic and clinical research in this area.

Biocompatibility of implant materials: a review and scanning electron microscopic study.

The evolution of various plastic materials used in the manufacture of intraocular lenses is reviewed, with special reference to the problem of biocompatibility of lens haptic materials. To date, polypropylene has been found and continues to be a highly effective, relatively inert material, providing good surgical results. However, the present study and other morphologic and chemical studies have provided evidence that indicates long-term alteration of polypropylene may occur. For this reason we believe that further studies of this particular polymer are warranted. Also, a continuing search and trial of other plastics to discover an equivalent or even more efficacious haptic material seems desirable. There is a need for careful long-term follow-up of patients after intraocular lens implantation, particularly of young patients.

Pathological and scanning electron microscopic evaluation of the 91Z intraocular lens.

We analyzed 18 explanted 91Z anterior chamber lenses by light and scanning electron microscopy. Intermittent touch of the lens loops to the posterior corneal surface and the anterior chamber angle may have occurred. Erosion of the polypropylene loops into the anterior chamber angle recess and into the iris tissue was also observed. Fibrous tissue, uveal tissue, and inflammatory debris were noted on the loops, forming dense synechias at the points of contact with the angle recess. In some lenses the edges of the optics were sharp. Other significant manufacturing defects were rarely seen, and there was no evidence of degradation of polypropylene loops. The problems regarding surgical removal of this lens are discussed.