Biomedical subjects

W John Armitage

Publications and source records attributed to W John Armitage.

8 recordsLinked to original sources

PubMed2005

Heart valve cryopreservation: protocol for addition of dimethyl sulphoxide and amelioration of putative amphotericin B toxicity.

AIM: To investigate the need for stepwise addition of dimethyl sulphoxide to heart valves and amelioration of putative amphotericin B toxicity. METHODS: There were four groups: an untreated control (Group 1) and three experimental groups. For the latter, porcine heart valves were exposed to the antibiotic/antimycotic mixture used for disinfecting heart valves in the Bristol Heart Valve Bank, for 24 h at 22 degrees C. Dimethyl sulphoxide (Me2SO, 10% v/v) was added either in two steps (5% then 10%) (Group 2) or in a single step. For single-step addition, valves were either first placed in Hanks' balanced salt solution for 10 min before transfer to the cryoprotectant solution (Group 3) or immersed directly in the 10% cryoprotectant solution (Group 4). The valve leaflets were dissected from the valves and frozen in 10% Me2SO in multi-well tissue culture plates at 1 degrees C/min to -80 degrees C. After storage overnight, the valve leaflets were warmed at approximately 11 degrees C/min and the cryoprotectant was removed by single-step dilution in excess Hartmann's solution. Each leaflet was then divided into four pieces, which were placed in separate wells of a culture plate. Outgrowth of cells from the explants was monitored daily and graded according to the extent of cell growth. RESULTS: After freezing and thawing, only 77% of the explants from valves placed directly into 10% Me2SO (Group 4) showed outgrowth of cells after freezing compared with 89% with two-step addition of Me2SO (Group 2) and 95% with one-step addition after the extra rinse in Hanks' solution (Group 3) (chi2, p=0.001). 92% of unfrozen control explants showed outgrowth of cells (Group 1). Only 37% of Group 4 explants reached confluence compared with 63 and 56%, respectively, of Groups 2 and 3 explants (chi2, p=0.007). The rates of cell growth in Group 2 (two-step addition of Me2SO) and Group 3 (one-step addition of Me2SO with additional Hanks' solution rinse) were similar and faster than the Group 4 (one-step addition of Me2SO without the additional Hanks' rinse). CONCLUSION: Single-step addition of Me2SO before freezing gave similar results to two-step addition provided an additional rinse in Hanks' solution was introduced after exposure to the antibiotic/antimycotic mixture. This suggests that antibiotic/antimycotic carryover may have been harmful during freezing and that the additional rinse in Hanks before one-step addition of Me2SO, and the 5% Me2SO step in the two-step protocol, merely served to reduce this carryover.

Amphotericin B↗

PubMed2004

Protocols for thawing and cryoprotectant dilution of heart valves.

PURPOSE: To reduce the time taken for thawing and removal of cryoprotectant from heart valves. METHODS: Three sets of experiments were carried out using porcine heart valves. The valves in all three experiments were first exposed to 10% (v/v) dimethyl sulphoxide (DMSO) by a 2-step protocol. Outcome was determined after the various experimental treatments by monitoring the outgrowth of cells from valve leaflet explants. Experiment 1-Dilution protocol. Valves exposed to 10% DMSO were subjected to 4-, 2- or 1-step dilution to remove the DMSO. Experiment 2-Warming rate. The rate of warming was increased by reducing the volume of cryoprotectant medium in which the valves were frozen. Valves were exposed to 10% DMSO, frozen in different volumes (100, 50, 25 or 0 ml) of cryoprotectant medium, and warmed in a 37 degrees C water bath. The DMSO was removed by 4-step dilution. Experiment 3-Standard vs. Modified protocol. Valves were either frozen in 100 ml 10% DMSO, thawed, and subjected to 4-step dilution (Standard) or frozen in 50 ml 10% DMSO, thawed, and the DMSO removed by single-step dilution (Modified). RESULTS: Neither the rate of warming nor the rate of dilution of DMSO had any influence on the subsequent outgrowth of valve leaflet fibroblasts. There were no differences in the outgrowth of cells from valve leaflets cryopreserved by the Standard or Modified protocols. CONCLUSION: The time taken for thawing and dilution of heart valves could be reduced from >20 min to <10 min without detriment to the viability of the leaflet fibroblasts. This should have a positive impact on valve replacement surgery as the thawing and dilution of valves are typically carried out while the patients are on cardiopulmonary bypass.

Animals↗

PubMed2004

Is directed donation misguided?

The worldwide shortage of organs and tissue for transplant has led to many ethical discussions involving restrictions concerning organ and tissue donation, including living donations and payment for donation. Efforts are being made to increase the donor pool; however, it is timely to ask whether moral decisions such as the rejection of directed donations are defensible. In this Clinical Conundrum, six specialists delve into the ethical and practical issues surrounding directed donation of human organs and tissues with particular reference to its implications in the field of ophthalmology.

Cornea↗

PubMed2003

Prospective, randomized clinical and endothelial evaluation of 2 storage times for cornea donor tissue in organ culture at 31 degrees C.

OBJECTIVE: To compare the endothelial and clinical outcome of penetrating keratoplasty with corneas stored in organ culture for up to 12 days (5-12 days; group 1) or more than 21 days (21-24 days; group 2). METHODS: We conducted a controlled double-masked trial. Storage durations were randomly assigned to the paired corneas, and endothelial cell density (ECD) was measured at the start and end of organ culture. Patients with a low rejection risk and preoperative ECD within the reference range were randomly assigned to 1 of the 2 groups and underwent an 8.25-mm penetrating keratoplasty (n = 25 pairs). Follow-up at day 5 and months 1, 6, and 12 included central ECD, morphometry, graft transparency, visual acuity, pachymetry, and complications. The main outcome measure was the central ECD at month 12. RESULTS: At the end of organ culture, ECD of the group 1 corneas was higher by 273 cells/mm2 (95% confidence interval [CI], 178-368; P<.001). One year after penetrating keratoplasty, the group 1 ECD was still comparably higher by 227 cells/mm2 (95% CI, 43-411; P =.02). Graft transparency, pachymetry, and complication rate did not differ at any time. In group 1, visual acuity was better at month 1. CONCLUSIONS: Shorter organ culture allows delivery of corneas with higher ECD. Recipients with ECD within the reference range and low rejection risk retain this initial benefit 1 year postoperatively. The higher endothelial cell capital may prevent or delay late endothelial failure, the leading cause of graft failure in these recipients. We therefore prefer short-term storage for such recipients.

Adult↗

PubMed2003

Cryopreservation of cornea: a low cooling rate improves functional survival of endothelium after freezing and thawing.

AIM: To investigate the influence of low cooling rates on endothelial function and morphology of corneas frozen with propane-1,2-diol (PROH). METHODS: Rabbit corneas, mounted on support rings, were exposed to 1.4mol/l (10% v/v) PROH, seeded to initiate freezing, and cooled at 0.2 or 1 degrees C/min to -80 degrees C. Corneas were frozen immersed in liquid or suspended in air. After being held overnight in liquid nitrogen, corneas were warmed at 1 or 20 degrees C/min. After stepwise removal of the cryoprotectant, the ability of the endothelium actively to control corneal hydration was monitored during normothermic perfusion. Morphology was assessed after staining with trypan blue and alizarin red S, and by specular microscopy during perfusion. RESULTS: Functional survival was achieved only after slow cooling (0.2 degrees C/min) with the cornea immersed in the cryoprotectant medium, and rapid warming (20 degrees C/min). These conditions also gave the best morphology after freezing and thawing. CONCLUSION: Cooling rates lower than those typically applied to cornea improved functional survival of the endothelium. This result is in accord with previous observations showing the benefit of low cooling rates for cell monolayers [CryoLetters 17 (1996) 213-218].

Animals↗

PubMed2003

Predicting endothelial cell loss and long-term corneal graft survival.

PURPOSE: To evaluate a biexponential decay model for describing the loss of corneal endothelial cells with age as well as the increased loss of cells after cataract surgery and penetrating keratoplasty. METHODS: Data from previous studies were identified and the sum of two exponentials, d = p. exp(-at) + q. exp(-bt) (where d is cell density at time t, p and q are constants the sum of which is equal to the initial cell density, and a and b are exponential rate constants), fitted to each data set by a nonlinear least-squares algorithm. Goodness of fit was indicated by the residual standard deviation. Half times were calculated from the exponential rate constants. RESULTS: The model identified in each instance a rapid and a slow component to the cell loss. The half time for the slow component of the loss with age was 224 years, underlining the excess endothelial capacity in normal eyes. After surgery, the rapid component of the cell loss was probably due to surgical trauma and, after penetrating keratoplasty, cell-mediated rejection and other complications. The half times of the slow component were only 26 years after cataract surgery and 21 years after penetrating keratoplasty. DISCUSSION: The loss of endothelial cells followed a biexponential decay and could thus be described by a single equation. The half times of the slow component of the cell loss after surgery were substantially less than for the loss with age, indicating a markedly increased rate of cell loss that persisted for many years after surgery. A mechanism for this accelerated cell loss is suggested that involves a nonspecific, innate response initiated by the breakdown of the blood-ocular barrier. The model was used to calculate endothelial cell loss in the long term after penetrating keratoplasty and to predict when cell density would reach levels that are incompatible with maintenance of transparency and graft function. Thus, a rationale is presented for the setting of minimum donor cell densities by eye banks.

Adolescent↗

PubMed2002

Recovery of endothelial function after vitrification of cornea at -110 degrees C.

PURPOSE: To determine whether endothelial function is retained after ice-free cryopreservation of cornea by vitrification at -110 degrees C. METHODS: Rabbit corneas, mounted on support rings, were exposed to a solution containing 6.8 M propane-1,2-diol (PROH) and cooled at approximately 7 degrees C/min to -110 degrees C, which was below the glass transition temperature (T(g)) of the solution. After rewarming at approximately 12 degrees C/min and removal of the PROH, endothelial function was assessed by monitoring corneal thickness during perfusion at 34 degrees C. RESULTS: Addition and removal of 6.8 M PROH without cooling to -110 degrees C did not markedly impair endothelial function, although corneas were thicker than control samples. There was no visible crystallization of ice during cooling to -110 degrees C; but a few small, discrete sites of crystallization remote from the endothelium, were observed during warming. After removal of the PROH, corneas approximately doubled in thickness during the first 3 hours of perfusion, but they then started to thin, which suggested active control of stromal hydration by the endothelium. This was confirmed in a further set of experiments by removal of bicarbonate ions from the perfusate at this point, which resulted in further swelling at +58 +/- 2 microm/hour (SD; n = 4). Restoring bicarbonate to the perfusate halted this swelling, and the corneas then thinned at -13 +/- 2 microm/hour (n = 4). Morphologically, staining with trypan blue and alizarin red S showed an apparently intact endothelial monolayer. CONCLUSIONS: Rabbit corneal endothelium tolerated exposure to 6.8 M PROH, and endothelial function was evident after vitrification at -110 degrees C. Preliminary morphologic results with vitrified human cornea also showed retention of endothelium.

Aged↗

PubMedDate not supplied

Transplantation of ocular tissue from a donor with sporadic Creutzfeldt-Jakob disease.

BACKGROUND: One definite, one probable and several possible transmissions of sporadic Creutzfeldt-Jakob disease (sCJD) have followed corneal transplantation. We report an incident in the UK in 1997 in which both corneas and scleras from a donor, subsequently confirmed to have had sCJD, were transplanted. The final clinical outcome for two surviving recipients is still not yet known. CASE REPORT: In 1997, a 56-year-old woman died from biopsy-proven carcinoma of the bronchus. Both eyes were donated for transplantation. Shortly before she died, she had developed neurological symptoms thought to be due to brain metastases. However, the final result of a neurological post-mortem examination revealed evidence of sCJD. By this time the corneas had been transplanted, one 3 months previously into a 40-year-old man for keratoconus and the other 4 months previously into an 85-year-old woman for Fuchs' dystrophy. In addition, both scleras had been transplanted into a 36-year-old man undergoing oculoplastic reconstructive surgery. The surgeons and patients were informed and removal of tissue was advised but undertaken in only two of the patients. Immunohistochemistry failed to demonstrate the presence of the abnormal form of the prion protein in explanted tissue. CONCLUSIONS: Eight years after the event, two patients remain free of symptoms suggestive of iatrogenic CJD (http://www.cjd.ed.ac.uk/criteria.htm). The third having died aged 92 years, some 7 years after surgery, showing signs of dementia not considered indicative of iatrogenic CJD. Nevertheless this adverse incident attracted substantial publicity. Coupled with continuing concerns in the UK about person-to-person transmission of variant CJD, this has lead to a number of important consequences in donor eye retrieval, ocular tissue banking and transplantation.

Adult↗