Inactivation of Bacillus anthracis and Bacillus atrophaeus spores on different surfaces with ultraviolet light produced with a low-pressure mercury vapor lamp or light emitting diodes.

AIMS: To obtain quantitative efficacy data of two ultraviolet light (UVC) technologies for surface inactivation of Bacillus anthracis Ames and Bacillus atrophaeus spores. METHODS AND RESULTS: Spores were deposited onto test coupons and controls of four different materials, via liquid suspension or aerosol deposition. The test coupons were then exposed to UVC light from either a low-pressure mercury vapor lamp or a system comprised of light emitting diodes, with a range of dosages. Positive controls were held at ambient conditions and not exposed to UVC light. Following exposure to UVC, spores were recovered from the coupons and efficacy was quantified in terms of log10 reduction (LR) in the number of viable spores compared to that from positive controls. CONCLUSIONS: Decontamination efficacy varied by material and UVC dosage (efficacy up to 5·7 LR was demonstrated). There was no statistical difference in efficacy between the two species or between inoculation methods. Efficacy improved for the LED lamp at lower relative humidity, but this effect was not observed with the mercury vapor lamp. SIGNIFICANCE AND IMPACT OF THE STUDY: This study will be useful in determining whether UVC could be used for the inactivation of B. anthracis spores on different surface types.

Heparin-protamine balance after neonatal cardiopulmonary bypass surgery.

Essentials Heparin-protamine balance (HPB) modulates bleeding after neonatal cardiopulmonary bypass (CPB). HPB was examined in 44 neonates undergoing CPB. Post-operative bleeding occurred in 36% and heparin rebound in 73%. Thrombin-initiated fibrin clot kinetic assay and partial thromboplastin time best assessed HPB. SUMMARY: Background Neonates undergoing cardiopulmonary bypass (CPB) are at risk of excessive bleeding. Blood is anticoagulated with heparin during CPB. Heparin activity is reversed with protamine at the end of CPB. Paradoxically, protamine also inhibits blood coagulation when it is dosed in excess of heparin. Objectives To evaluate heparin-protamine balance in neonates undergoing CPB by using research and clinical assays, and to determine its association with postoperative bleeding. Patients/Methods Neonates undergoing CPB in the first 30 days of life were studied. Blood samples were obtained during and after surgery. Heparin-protamine balance was assessed with calibrated automated thrombography, thrombin-initiated fibrin clot kinetic assay (TFCK), activated partial thromboplastin time (APTT), anti-FXa activity, and thromboelastometry. Excessive postoperative bleeding was determined by measurement of chest tube output or the development of cardiac tamponade. Results and Conclusions Of 44 neonates enrolled, 16 (36%) had excessive postoperative bleeding. The TFCK value was increased. By heparin in neonatal blood samples, but was only minimally altered by excess protamine. Therefore, it reliably measured heparin in samples containing a wide range of heparin and protamine concentrations. The APTT most closely correlated with TFCK results, whereas anti-FXa and thromboelastometry assays were less correlative. The TFCK and APTT assay also consistently detected postoperative heparin rebound, providing an important continued role for these long-established coagulation tests in the management of postoperative bleeding in neonates requiring cardiac surgical repair. None of the coagulation tests predicted the neonates who experienced postoperative bleeding, reflecting the multifactorial causes of bleeding in this population.

HIF signalling: The eyes have it.

The hypoxia inducible factors (HIFs) promote changes in gene expression in response to hypoxia, and mediate key physiological responses such as angiogenesis. They play important roles in development and normal physiology, as well as in ischaemic and other pathologies. The human eye is a complex organ, with tight regulation of vascularisation and oxygen delivery, with the highly specialised retina containing both highly vascularised and avascular regions. This review, written to honour the significant contribution of Lorenz Poellinger to this field, covers the role of the HIFs in normal development of the eye, specifically the vasculature, as well as their roles in numerous retinal pathologies, including ischaemic retinopathies, and age-related macular degeneration (AMD). The characterisation of the HIFs in the eye has improved our understanding of the development, function, and numerous pathologies of the eye, and should inform future therapeutic approaches.

A simple decontamination approach using hydrogen peroxide vapour for Bacillus anthracis spore inactivation.

AIMS: To evaluate the use of relatively low levels of hydrogen peroxide vapour (HPV) for the inactivation of Bacillus anthracis spores within an indoor environment. METHODS AND RESULTS: Laboratory-scale decontamination tests were conducted using bacterial spores of both B. anthracis Ames and Bacillus atrophaeus inoculated onto several types of materials. Pilot-scale tests were also conducted using a larger chamber furnished as an indoor office. Commercial off-the-shelf (COTS) humidifiers filled with aqueous solutions of 3 or 8% hydrogen peroxide (H2 O2 ) were used to generate the HPV inside the mock office. The spores were exposed to HPV for periods ranging from 8 h up to 1 week. CONCLUSIONS: Four- to seven-day exposures to low levels of HPV (average air concentrations of approx. 5-10 parts per million) were effective in inactivating B. anthracis spores on multiple materials. The HPV can be generated with COTS humidifiers and household H2 O2 solutions. With the exception of one test/material, B. atrophaeus spores were equally or more resistant to HPV inactivation compared to those from B. anthracis Ames. SIGNIFICANCE AND IMPACT OF THE STUDY: This simple and effective decontamination method is another option that could be widely applied in the event of a B. anthracis spore release.

Whole-building decontamination of Bacillus anthracis Sterne spores by methyl bromide fumigation.

AIMS: To evaluate the field inactivation of Bacillus anthracis Sterne spores with methyl bromide (MB) using commercial fumigation techniques. METHODS AND RESULTS: Eighty-seven wood and 87 glass coupons each containing ca. 1 × 10(6) B. anthracis Sterne spores, were placed in 22 locations inside a 1444 m(3) conference building. Four additional 12-coupon sets (six wood, six glass) were removed from the building at 16, 24, 32 and 40 h during fumigation. The building was sealed under two tarpaulins and fumigated with MB at ≥225 g m(-3) mean concentration for 48 h at 28°C and 83% RH. All B. anthracis spores fumigated for more than 16 h were inactivated. A single wood coupon from the 16-h set yielded ca. 2 × 10(3)  CFU. No damage to the building or its contents was observed. CONCLUSIONS: MB fumigation is a rapid, economical and effective whole-structure decontamination method for B. anthracis spores. SIGNIFICANCE AND IMPACT OF THE STUDY: MB fumigation offers a method of whole-structure B. anthracis decontamination without removal of materials, damage to sensitive electronics, costly indoor retrofitting.

Fumigation of a laboratory-scale HVAC system with hydrogen peroxide for decontamination following a biological contamination incident.

AIMS: To evaluate hydrogen peroxide vapour (H2 O2 ) for its ability to inactivate Bacillus spores within a laboratory-scale heating, ventilation and air-conditioning (HVAC) duct system. METHODS AND RESULTS: Experiments were conducted in a closed-loop duct system, constructed of either internally lined or unlined galvanized metal. Bacterial spores were aerosol-deposited onto 18-mm-diameter test material coupons and strategically placed at several locations within the duct environment. Various concentrations of H2 O2 and exposure times were evaluated to determine the sporicidal efficacy and minimum exposure needed for decontamination. For the unlined duct, high variability was observed in the recovery of spores between sample locations, likely due to complex, unpredictable flow patterns within the ducts. In comparison, the lined duct exhibited a significant desorption of the H2 O2 following the fumigant dwell period and thus resulted in complete decontamination at all sampling locations. CONCLUSIONS: These findings suggest that decontamination of Bacillus spore-contaminated unlined HVAC ducts by hydrogen peroxide fumigation may require more stringent conditions (higher concentrations, longer dwell duration) than internally insulated ductwork. SIGNIFICANCE AND IMPACT OF THE STUDY: These data may help emergency responders when developing remediation plans during building decontamination.

Environmental persistence of vaccinia virus on materials.

Smallpox is caused by the variola virus, and ranks as one of the most serious diseases that could originate from a biological weapon. However, limited data exist on the persistence of variola and related viruses on materials (that may act as fomites), under controlled environmental conditions. To fill these data gaps, we determined the persistence of the vaccinia virus (an established surrogate for the variola virus) as a function of temperature, relative humidity and material. Experiments were conducted with vaccinia virus in a freeze-dried form, using four materials under four sets of environmental conditions. After elapsed times ranging from 1 to 56 days, the virus was extracted from small coupons and quantified via plaque-forming units (PFU). The vaccinia virus was most persistent at low temperature and low relative humidity, with greater than 10(4) PFU recovered from glass, galvanized steel and painted cinder block at 56 days (equivalent to only a c. 2 log reduction). Thus, vaccinia virus may persist from weeks to months, depending on the material and environmental conditions. This study may aid those responsible for infection control to make informed decisions regarding the need for environmental decontamination following the release of an agent such as variola.

Comparison of the inhibitory activities of human tissue factor pathway inhibitor (TFPI)α and TFPIß.

BACKGROUND: Tissue factor pathway inhibitor (TFPI) is an alternatively spliced protein with two isoforms, TFPIα and TFPIß, which differ in their C-terminal structure and cellular localization. Detailed characterization of their inhibitory activity is needed to define potentially unique inhibitory roles in tissue factor (TF)-mediated thrombotic and inflammatory disease, and to understand how pharmaceuticals targeted to different structural regions of the TFPI isoforms alter hemostasis in hemophilia patients. METHODS: The TF inhibitory activity of TFPIß localized to the surface of CHO cells was compared with that of soluble TFPIα by the use of in vitro and in vivo assays. RESULTS: In TF-factor VIIa-mediated FXa generation assays, TFPIß was a slightly better inhibitor than TFPIα, which was approximately three-fold better than TFPI-160, a soluble, altered form of TFPI similar to TFPIß. In direct FXa inhibitory assays, TFPIß had an IC50 2.5-fold lower than that of TFPIα and 56-fold lower than that of TFPI-160. TFPIß inhibited TF-mediated CHO cell migration though Matrigel, whereas TFPIα and TFPI-160 were poor inhibitors, demonstrating that TFPIß effectively blocks TF-initiated signaling events during cellular migration through matrices that are not permeable to soluble forms of TFPI. Furthermore, TFPIß inhibited TF-dependent CHO cell infiltration into lung tissue following tail vein injection into SCID mice, and blocked the development of consumptive coagulopathy. CONCLUSIONS: TFPIß is a slightly better inhibitor of TF procoagulant activity than TFPIα. As a surface-associated protein, TFPIß is a much better inhibitor of TF-mediated cellular migration than soluble TFPIα, and may specifically act in the inhibition of TF-mediated signaling events on inflamed endothelium and/or monocytes.

Laboratory evaluation of large-scale decontamination approaches.

AIMS: To evaluate the effectiveness of two spray-based decontamination methods for surface contamination reduction and to determine the potential for contamination spread by these methods. METHODS AND RESULTS: Material coupons (treated plywood and concrete) were contaminated with c. 1 × 10(7) spores of Bacillus atrophaeus by aerosol deposition. Decontaminants (pH-adjusted bleach or Spor-Klenz(®) RTU) were applied to coupons by either backpack sprayer or gas-powered sprayer. Contact time, reapplication frequency and rinse method were also varied. In addition to surface removal efficacy, partitioning of contamination between the rinsate and aerosol fractions was determined. Results indicated that pH-adjusted bleach was effective (≥6 logs reduction) when two applications and a 30 min contact time were administered, regardless of the decontaminant application method or material. Spor-Klenz(®) RTU was effective on wood, but achieved ≤3 logs reduction on concrete. A shortened application procedure with pH-adjusted bleach resulted in lower efficacy on wood, and a greater apparent potential for contamination spread. CONCLUSIONS: Consideration of material surface type is important when selecting a decontaminant. Also, achieving conditions that effectively inactivate surface biological contamination are critical to preventing the spread of contamination. SIGNIFICANCE AND IMPACT OF THE STUDY: Results presented here are intended to help development of remediation plans following a biological contamination incident.

Optimizing acidified bleach solutions to improve sporicidal efficacy on building materials.

AIMS: We evaluated whether lowering pH (with acetic acid) and raising free available chlorine (FAC) levels in bleach solutions would improve efficacy in inactivating Bacillus spores on different materials. We also determined how varying pH and FAC levels affected bleach stability. METHODS AND RESULTS: Acidified bleach solutions with pH levels of 4.5, 6 and 7.5 and FAC levels between 5000 and 10,000 ppm were evaluated for decontamination efficacy against Bacillus subtilis spores inoculated onto test coupons made from wood, ceramic and galvanized steel. Lowering the pH or increasing the FAC level improved efficacy in some of the tests, but depended on the material, which significantly affected decontamination efficacy. The acidified bleach at pH of 7.5 was significantly less effective than bleach at a pH of 4.5 or 6. The FAC levels in the bleach were the most stable at pH 4.5, and stability at pH 4.5 was not significantly affected by the initial FAC level. CONCLUSIONS: It may be advisable to use bleach solutions with lower pH (rather than high FAC levels) in light of both the decontamination efficacy and bleach stability results. For wood materials, use of sporicides other than acidified bleach may be warranted. SIGNIFICANCE AND IMPACT OF THE STUDY: These results may be useful in preparing acidified bleach solutions for decontamination of materials contaminated with spores such as Bacillus anthracis.

Efficacy of liquid spray decontaminants for inactivation of Bacillus anthracis spores on building and outdoor materials.

AIMS: To obtain data on the efficacy of various liquid and foam decontamination technologies to inactivate Bacillus anthracis Ames and Bacillus subtilis spores on building and outdoor materials. METHODS AND RESULTS: Spores were inoculated onto test coupons and positive control coupons of nine different materials. Six different sporicidal liquids were spray-applied to the test coupons and remained in contact for exposure times ranging from 10 to 70 min. Following decontamination, spores were recovered from the coupons and efficacy was quantified in terms of log reduction. CONCLUSIONS: The hydrogen peroxide/peracetic acid products were the most effective, followed by decontaminants utilizing hypochlorous acid chemistry. Decontamination efficacy varied by material type. SIGNIFICANCE AND IMPACT OF THE STUDY: The study results may be useful in the selection of technologies to decontaminate buildings and outdoor areas in the event of contamination with B. anthracis spores. These results may also facilitate selection of decontaminant liquids for the inactivation of other spore-forming infectious disease agents.

Dry thermal resistance of Bacillus anthracis (Sterne) spores and spores of other Bacillus species: implications for biological agent destruction via waste incineration.

AIMS: To obtain needed data on the dry thermal resistance of Bacillus anthracis spores and other Bacillus species for waste incinerator applications. METHODS AND RESULTS: Tests were conducted in a pilot-scale incinerator utilizing biological indicators comprised of spores of Geobacillus stearothermophilus, Bacillus atrophaeus and B. anthracis (Sterne) and embedded in building material bundles. Tests were also conducted in a dry heat oven to determine the destruction kinetics for the same species. In the pilot-scale incinerator tests, B. atrophaeus and G. stearothermophilus demonstrated similar thermal sensitivity, but B. anthracis (Sterne) was less thermally resistant than G. stearothermophilus. For the dry heat oven tests conducted at 175°C, the D-values were 0·4, 0·2 and 0·3 min for B. atrophaeus, B. anthracis (Sterne) and G. stearothermophilus, respectively. CONCLUSIONS: Bacillus anthracis (Sterne) possesses similar or less dry heat resistance compared to B. atrophaeus and G. stearothermophilus. SIGNIFICANCE AND IMPACT OF THE STUDY: Previous studies have demonstrated conditions under which bacterial spores may survive in an incinerator environment. The data from this study may assist in the selection of surrogates or indicator micro-organisms to ensure B. anthracis spores embedded in building materials are completely inactivated in an incinerator.

A hypothesis to suggest that light is a risk factor in glaucoma and the mitochondrial optic neuropathies.

The authors propose that light entering the eye interacts with retinal ganglion cell (RGC) axon mitochondria to generate reactive oxygen intermediates (ROI) and that when these neurons are in an energetically low state, their capacity to remove these damaging molecules is exceeded and their survival is compromised. They suggest that in the initial stages of glaucoma, RGCs exist at a low energy level because of a reduced blood flow at the optic nerve head and that in the mitochondrial optic neuropathies (MONs), this results from a primary, genetic defect in aerobic metabolism. In these states RGCs function at a reduced energy level and incident light on the retina becomes a risk factor. Preliminary laboratory studies support this proposition. Firstly, the authors have shown that light is detrimental to isolated mitochondria in an intensity dependent manner. Secondly, light triggers apoptosis of cultured, transformed RGCs and this effect is exacerbated when the cells are nutritionally deprived. Detailed studies are under way to strengthen the proposed theory. On the basis of this proposal, the authors suggest that patients with optic neuropathies such as glaucoma or at risk of developing a MON may benefit from the use of spectral filters and reducing the intensity of light entering the eye.

Neuronal death in primary retinal cultures is related to nitric oxide production, and is inhibited by erythropoietin in a glucose-sensitive manner.

The aim of this work was to investigate the interrelated effects of glucose, nitric oxide (NO) and erythropoietin on neuronal survival in retinal cultures, thereby exploring the mechanism of neuronal death in the diabetic retina. Rat retinal cells were cultured in low (5 mM) or high (15 mM) glucose concentrations. After 9 days, cell viability was assessed by (3,4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and NO production was determined by the Griess reaction. Immunohistochemistry was used to quantify GABA-labelled neurones and cells staining for DNA breakdown. High or low glucose concentrations had no effect on basal NO production or the survival of neurones in culture, but treatment with N-nitro-L-arginine methyl ester reduced extracellular levels of NO and increased neuronal survival at both concentrations of glucose. Erythropoietin decreased cell death and NO levels, but only in cultures grown in low concentrations of glucose. It is concluded that erythropoietin's neurotrophic function in the retina is attenuated at glucose concentrations similar to those which occur in diabetes.

Optic nerve and neuroprotection strategies.

BACKGROUND: Experimental studies have yielded a wealth of information related to the mechanism of ganglion cell death following injury either to the myelinated ganglion cell axon or to the ganglion cell body. However, no suitable animal models exist where injury can be directed to the optic nerve head region, particularly the unmyelinated ganglion cell axons. The process of relating the data from the various animal models to many different types of optic neuropathies in man must, therefore, be cautious. RESULTS: Extensive studies on the isolated optic nerve have yielded valuable information on the way white matter is affected by ischaemia and how certain types of compounds can attenuate the process. Moreover, there are now persuasive data on how ganglion cell survival is affected when the ocular blood flow is reduced in various animal models. As a consequence, the molecular mechanisms involved in ganglion cell death are fairly well understood and various pharmacological agents have been shown to blunt the process when delivered before or shortly after the insult. CONCLUSIONS: A battery of agents now exist that can blunt animal ganglion cell death irrespective of whether the insult was to the ganglion cell body or the myelinated axon. Whether this information can be applied for use in patients remains a matter of debate, and major obstacles need to be overcome before the laboratory studies may be applied clinically. These include the delivery of the pharmacological agents to the site of ganglion cell injury and side effects to the patients. Moreover, it is necessary to establish whether effective neuroprotection is only possible when the drug is administered at a defined time after injury to the ganglion cells. This information is essential in order to pursue the idea that a neuroprotective strategy can be applied to a disease like glaucoma, where ganglion cell death appears to occur at different times during the lifetime of the patient.

Methamphetamine exacerbates the toxic effect of kainic acid in the adult rat retina.

The recreational use of the psychoactive drug, methamphetamine has increased markedly over the last three decades. It has long been known that this drug has detrimental effects upon the mammalian brain monoaminergic system, but the long- or short-term effects on the retina, a neurological extension of the central nervous system, have received little attention. The aim of this study was, therefore, to determine whether intraocular injection of methamphetamine (MA) is toxic to the healthy adult rat retina and to analyse its effects on the compromised retina after an injection of the ionotropic glutamate receptor agonist, kainate, which is known to cause retinal neuropathology. The equivalent of 1 mM (in the vitreous humour) MA and/or kainate (40 microM) were injected intravitreally. Flash electroretinograms (ERGs) were recorded before and 2 and 4 days after treatment. Five days after treatment, animals were killed and the retinas analysed either for the immunohistochemical localisation of various antigens or for electrophoresis/Western blotting. Some animals were kept for 19 days after treatment and the retinas analysed for tyrosine hydroxylase immunoreactivity. No differences could be found between vehicle- and MA-treated retinas with respect to the nature or localisation of either tyrosine hydroxylase immunoreactivity after 5 or 19 days or other antigens after 5 days. Moreover, the normal ERG and GFAP and calretinin protein antigens were unaffected by MA. Kainate treatment, however, caused a change in the ERGs after 2 and 4 days, an alteration in every antigen localised by immunohistochemistry and an increase in the retinal levels of calretinin and GFAP proteins. Significantly, the changes seen in the b-wave amplitude and implicit time of the ERG after 4 days and the increased level of GFAP protein after 5 days following kainate treatment were enhanced when MA was co-injected. Intravitreal injection of methamphetamine had no detectable detrimental effect on the normal adult rat retina but exacerbated the damaging effects of kainic acid. Such data suggest that a neurotoxic effect of MA may be more obviously illustrated when the tissue is already compromised as occurs in, for example, ischemia.

Hypoglycaemia exacerbates ischaemic retinal injury in rats.

AIMS: To determine the effect of hypoglycaemia on ischaemic retinal injury. METHODS: Rat retinal cultures were incubated in varying concentrations of glucose while placed under standardised anoxic conditions, and the number of surviving GABA immunoreactive neurons was assessed using immunocytochemistry. Hypoglycaemia was induced in age and sex matched Wistar rats by an injection of rapid acting insulin. The blood, vitreous, and retinal glucose concentrations were measured using a hexokinase assay kit. Electroretinography, semiquantitative RT-PCR, and histology were used to compare the functional and structural retinal injury in these rats with the injury in appropriate controls after a period of pressure induced retinal ischaemia. RESULTS: Retinal cultures maintained in low glucose concentrations (<1 mM) had fewer surviving GABA immunoreactive neurons after an anoxic insult compared with retinal cultures maintained in 5 mM glucose. Hypoglycaemic rats had significantly lower vitreous glucose concentrations (0.57 (SEM 0.04) mM) than the control rats (3.1 (0.70) mM; p<0.001). The a-wave and b-wave amplitudes of the hypoglycaemic rats after 3 and 7 days of reperfusion were significantly lower than the amplitudes of the control rats. Furthermore, the level of Thy-1 mRNA (a retinal ganglion cell marker) was significantly lower in the hypoglycaemic group (p<0.001) and there was a corresponding exacerbation of structural injury compared with the controls. CONCLUSION: Hypoglycaemia causes a significant reduction in vitreous glucose levels and exacerbates ischaemic retinal injury.

Effectiveness of levobetaxolol and timolol at blunting retinal ischaemia is related to their calcium and sodium blocking activities: relevance to glaucoma.

Glaucoma is a chronic optic neuropathy in which retinal ganglion cells die over a number of years. The initiation of the disease and its progression may involve an ischaemic-like insult to the ganglion cell axons caused by an alteration in the quality of blood flow. Thus, to effectively treat glaucoma it may be necessary to counteract the ischaemic-like insult to the region of the optic nerve head. Studies on the isolated optic nerve suggest that substances that reduce the influx of sodium would be particularly effective neuroprotectants. Significantly, of the presently used antiglaucoma substances, only beta-blockers can reduce sodium influx into cells. Moreover, they also reduce the influx of calcium and this would be expected to benefit the survival of insulted neurones. Betaxolol is the most effective antiglaucoma drug at reducing sodium/calcium influx. Our electroretinographic data indicated that topical application of levobetaxolol to rats attenuated the effects of ischaemia/reperfusion injury. Timolol was also effective but to a lesser extent. Based on these data we conclude that beta-blockers may be able to blunt ganglion cell death in glaucoma, and that levobetaxolol may be a more effective neuroprotectant than timolol because of its greater capacity to block sodium and calcium influx.

The beta-adrenoceptor antagonists metipranolol and timolol are retinal neuroprotectants: comparison with betaxolol.

beta-adrenoceptor antagonists are used clinically to reduce elevated intraocular pressure in glaucoma which is characterised by a loss of retinal ganglion cells. Previous studies have shown that the beta(1)-selective adrenoceptor antagonist, betaxolol, is additionally able to protect retinal neurones in vitro and ganglion cells in vivo from the detrimental effects of either ischemia-reperfusion or from excitotoxicity, after topical application. The neuroprotective effect of betaxolol is thought not to be elicited through an interaction with beta-adrenoceptors, but by its ability to reduce influx of sodium and calcium through voltage-sensitive calcium and sodium channels. In the present study it is shown that the non-selective beta-adrenoceptor antagonists, metipranolol and timolol behave like betaxolol. When topically applied they all attenuate the detrimental effect of ischemia-reperfusion. Protection of the retina was determined by evaluating changes in the electroretinogram and by assessing the loss of mRNA for Thy-1, which is expressed in retinal ganglion cells. In addition, studies conducted on neurones in mixed retinal cultures demonstrated that metipranolol, betaxolol and timolol were all able to partially counteract anoxia-induced cell loss and viability reduction. The influence of timolol was, however, not significant. Within the confines of these investigations, an order of neuroprotective efficacy was delineated for the three beta-adrenoceptor antagonists: betaxolol>metipranolol>timolol. The ability of the beta-adrenoceptor antagonists to attenuate ligand-induced stimulation of calcium and sodium entry into neuronal preparations showed a similar order of effectiveness. In conclusion, the ability to confer neuroprotection to retinal neurones is a common feature of three ophthalmic beta-adrenoceptor antagonists (betaxolol, metipranolol and timolol). A comparison of the effectiveness of the individual compounds in protecting retinal cells in vivo was not possible in these studies. However, in vitro studies show that the capacity of the individual beta-adrenoceptor antagonists to act as neuroprotectants appears to relate to their capacity to attenuate neuronal calcium and sodium influx.