Inability to work following COVID-19 vaccination-a relevant aspect for future booster vaccinations.

OBJECTIVES: COVID-19 vaccination is a key prevention strategy to reduce the spread and severity of SARS-CoV-2 infections. However, vaccine-related inability to work among healthcare workers (HCWs) could overstrain healthcare systems. STUDY DESIGN: The study presented was conducted as part of the prospective CoVacSer cohort study. METHODS: This study examined sick leave and intake of pro re nata medication after the first, second, and third COVID-19 vaccination in HCWs. Data were collected by using an electronic questionnaire. RESULTS: Among 1704 HCWs enrolled, 595 (34.9%) HCWs were on sick leave following at least one COVID-19 vaccination, leading to a total number of 1550 sick days. Both the absolute sick days and the rate of HCWs on sick leave significantly increased with each subsequent vaccination. Comparing BNT162b2mRNA and mRNA-1273, the difference in sick leave was not significant after the second dose, but mRNA-1273 induced a significantly longer and more frequent sick leave after the third. CONCLUSION: In the light of further COVID-19 infection waves and booster vaccinations, there is a risk of additional staff shortages due to postvaccination inability to work, which could negatively impact the already strained healthcare system and jeopardise patient care. These findings will aid further vaccination campaigns to minimise the impact of staff absences on the healthcare system.

Piloting a partially self-financed mode of human immunodeficiency virus pre-exposure prophylaxis delivery for men who have sex with men in Hong Kong.

INTRODUCTION: Pre-exposure prophylaxis (PrEP) with tenofovir disoproxil fumarate (TDF) 300 mg/emtricitabine (FTC) 200 mg is a proven strategy for preventing human immunodeficiency virus (HIV) transmission in men who have sex with men (MSM). This study aimed to test the feasibility and acceptability of PrEP delivered at a pilot clinic for MSM in Hong Kong, where PrEP service is currently unavailable. METHODS: Partially self-financed PrEP was provided to HIV-negative adult MSM with high behavioural risk of HIV transmission after excluding hepatitis B infection and renal insufficiency. Participants received daily TDF/FTC for 30 weeks at 13.3% of the drug cost. Adherence and behaviours were monitored through questionnaires while creatinine and HIV/STI (sexually transmitted infection) incidence were monitored with point-of-care and laboratory tests. Preference for continuing with PrEP was evaluated at the end of the prescription period. RESULTS: Seventy-one PrEP-naïve MSM were included in the study, of whom 57 (80%) were retained at the end of 28 weeks. Satisfactory adherence and self-limiting adverse events were reported, while none of the participants contracted HIV. Risk compensation was observed, with an STI incidence of 3.17 per 100 person-years. At the end of the prescription period, a majority (89%) indicated interest in continuing with PrEP. Preference for PrEP was associated with age ≥28 years and peer influence (P=0.04), while stigma was a concern. Price was a deterrent to self-financed PrEP, and only half (51%) considered a monthly cost of ≤HK$500 (US$1=HK$7.8) as reasonable. CONCLUSIONS: A partially self-financed mode of PrEP delivery is feasible with good retention in MSM in Hong Kong.

Analysis of non-typeable Haemophilus influenzae in invasive disease reveals lack of the capsule locus.

Among invasive Haemophilus influenzae, unencapsulated strains have largely surpassed the previously predominant serotype b (Hib) because of Hib vaccination. Isolates without the genomic capsule (cap) locus are designated non-typeable H. influenzae (NTHi). They are different from capsule-deficient variants that show deletion of the capsule transport gene bexA within the cap locus. The frequency of capsule-deficient variants in invasive disease is unknown. We analysed 783 unencapsulated invasive isolates collected over 5 years in Germany and found no single capsule-deficient isolate. Invasive unencapsulated strains in Germany were exclusively NTHi. A negative serotyping result by slide agglutination was therefore highly predictive for NTHi.

Rapid hippocampal network adaptation to recurring synchronous activity--a role for calcineurin.

Neuronal networks are thought to gradually adapt to altered neuronal activity over many hours and days. For instance, when activity is increased by suppressing synaptic inhibition, excitatory synaptic transmission is reduced. The underlying compensatory cellular and molecular mechanisms are thought to contribute in important ways to maintaining normal network operations. Seizures, due to their massive and highly synchronised discharging, probably challenge the adaptive properties of neurons, especially when seizures are frequent and intense - a condition common in early childhood. In the experiments reported here, we used rat and mice hippocampal slice cultures to explore the effects that recurring seizure-like activity has on the developing hippocampus. We found that developing networks adapted rapidly to recurring synchronised activity in that the duration of seizure-like events was reduced by 42% after 4 h of activity. At the same time, the frequency of spontaneous excitatory postsynaptic currents in pyramidal cells, the expression of biochemical biomarkers for glutamatergic synapses and the branching of pyramidal cell dendrites were all dramatically reduced. Experiments also showed that the reduction in N-methyl-D-aspartate receptor subunits and postsynaptic density protein 95 expression were N-methyl-D-aspartate receptor-dependent. To explore calcium signaling mechanisms in network adaptation, we tested inhibitors of calcineurin, a protein phosphatase known to play roles in synaptic plasticity and activity-dependent dendrite remodeling. We found that FK506 was able to prevent all of the electrophysiological, biochemical, and anatomical changes produced by synchronised network activity. Our results show that hippocampal pyramidal cells and their networks adapt rapidly to intense synchronised activity and that calcineurin play an important role in the underlying processes.

The evolutionary dynamics of influenza A virus adaptation to mammalian hosts.

Few questions on infectious disease are more important than understanding how and why avian influenza A viruses successfully emerge in mammalian populations, yet little is known about the rate and nature of the virus' genetic adaptation in new hosts. Here, we measure, for the first time, the genomic rate of adaptive evolution of swine influenza viruses (SwIV) that originated in birds. By using a curated dataset of more than 24 000 human and swine influenza gene sequences, including 41 newly characterized genomes, we reconstructed the adaptive dynamics of three major SwIV lineages (Eurasian, EA; classical swine, CS; triple reassortant, TR). We found that, following the transfer of the EA lineage from birds to swine in the late 1970s, EA virus genes have undergone substantially faster adaptive evolution than those of the CS lineage, which had circulated among swine for decades. Further, the adaptation rates of the EA lineage antigenic haemagglutinin and neuraminidase genes were unexpectedly high and similar to those observed in human influenza A. We show that the successful establishment of avian influenza viruses in swine is associated with raised adaptive evolution across the entire genome for many years after zoonosis, reflecting the contribution of multiple mutations to the coordinated optimization of viral fitness in a new environment. This dynamics is replicated independently in the polymerase genes of the TR lineage, which established in swine following separate transmission from non-swine hosts.

[Infections with pneumococci, menigococci, H. influenzae and diphtheria in Germany: the RKI Reference Network for Invasive Bacterial Infections (IBI) at the 5th Würzburg Workshop on Epidemiology, Prevention and Therapy for Invasive Meningococcal Diseases 2010 (meeting report)]. / Infektionen mit Pneumokokken, Meningokokken, H. influenzae und Diphtherie in Deutschland: das RKI Referenznetzwerk für Invasive Bakterielle Infektionen (IBI) beim 5. Würzburger Workshop zur Epidemiologie, Prävention und Therapie von invasiven Meningokokkenerkrankung 2010 (Tagungsbericht).

The surveillance and prevention of invasive bacterial infections requires flexible strategic coordination of all involved health-care professionals. For this purpose, the German National Reference Centres for Meningococci, Streptococci and the Consultant Laboratories for Haemophilus influenzae and diphtheria have formed the Reference Network for Invasive bacterial infections (IBI). The 5th Würzburg Workshop on Meningococcal Diseases 2010 provided the network with a forum for the interdisciplinary exchange between scientists, public health professionals, medical microbiologists and clinicians. The topics covered the analysis of surveillance data for meningococcal disease in the last decade, as well as methods to control for antibody response following vaccination, including a serum bactericidal antibody (SBA) assay, and the development of new vaccines that also include the most common serogroup B. The presentation on diphtheria showed that this rare disease in Germany has become a diagnostic challenge, and that apart from the classical pathogen also toxigenic C. ulcerans strains must be considered. Due to the successful vaccination against Hib, H. influenzae disease has changed from a classical childhood disease to an infection of elderly people mainly caused by unencapsulated strains. Following the introduction of vaccines, changes in the serotype distribution and antibiotic resistance profiles have become apparent for S. pneumoniae infections. The epidemiological data were complemented by clinical aspects concerning the vaccination of immunocompromised children.

Evaluation of the VITEK 2 AST-N111 card for detection of extended-spectrum beta-lactamases (ESBLs) in Escherichia coli, Klebsiella pneumoniae, and Klebsiella oxytoca compared to ESBL Etests and combination disk methods.

The VITEK 2 AST-N111 card was evaluated for detection of extended-spectrum beta-lactamases (ESBLs) by testing 51 ESBL positive and 50 ESBL negative isolates of E. coli, K. pneumoniae, and K. oxytoca. The occurrence of beta-lactamase genes was confirmed by PCR and sequencing. The advanced expert system (AES) of the VITEK 2 system achieved sensitivity and specificity values of 100% and 96.0%, respectively. The ESBL test of the VITEK 2 AST-N111 card showed a sensitivity of 92.1% and a specificity of 90.0%. Contradictory results obtained with the two VITEK 2 tools could be clarified by combination disk tests in nine of 11 isolates. The combined use of AES and ESBL tests of the AST-N111 card in association with combination disk tests in case of contradictory results seems to be a reliable method for ESBL detection.

Palmitoylation of nicotinic acetylcholine receptors.

It is well established that nicotinic acetylcholine receptors (nAChRs) undergo a number of different posttranslational modifications, such as disulfide bond formation, glycosylation, and phosphorylation. Recently, our laboratory has developed more sensitive assays of protein palmitoylation that have allowed us and others to detect the palmitoylation of relatively low abundant proteins such as ligand-gated ion channels. Here, we present evidence that palmitoylation is prevalent on many subunits of different nAChR subtypes, both muscle-type nAChRs and the neuronal "alpha(4)beta(2)" and "alpha(7)" subtypes most abundant in brain. The loss of ligand binding sites that occurs when palmitoylation is blocked with the inhibitor bromopalmitate suggests that palmitoylation of alpha(4)beta(2) and alpha(7) subtypes occurs during subunit assembly and regulates the formation of ligand binding sites. However, additional experiments are needed to test whether nAChR subunit palmitoylation is involved in other aspects of nAChR trafficking or whether palmitoylation regulates nAChR function. Further investigation would be aided by identifying the sites of palmitoylation on the subunits, and here we propose a mass spectrometry strategy for identification of these sites.

Use of echocardiography to assess function of hDAF-transgenic pig cardiac xenografts.

The current standard of hand palpation may not be a sensitive method to detect rejection in heterotopic heart xenotransplants (HHTx). We sought to assess the use of echocardiography to detect rejection of pig heart xenografts. Four cynomolgus monkeys received HHTx from hDAF-transgenic pigs. Immunosuppression was cyclophosphamide induction, cyclosporine, steroids, sodium mycophenolate, alphaGal trisaccharide polymer, +/-soluble complement receptor type 1. Echocardiography was performed immediately after HHTx and three times a week postoperatively. Contractility on echo was scored as 1(none), 2(severely impaired), 3(moderate to severely impaired), 4(moderately impaired), 5(mild to moderately impaired), 6(mildly impaired), or 7(normal). Left ventricle wall thickness (LVWT) was measured in the anterior, inferior, posterior, lateral, and septal walls, the average was calculated. Impaired contractility or increase in LVWT were considered rejection and treated with steroids (solumedrol 15 mg/kg IV for 3-5 days). Palpation score (4-strong to 1-none) was recorded daily. Myocardial biopsies were obtained infrequently. At the time of first rejection, all four monkeys had an increase in LVWT and a decrease in contractility on echocardiography. Steroid treatment enhanced contractility in four monkeys and decreased LVWT in three monkeys. Palpation score remained at four of four during initial rejection episodes. Decrease in contractility and increase in LVWT on echocardiography appear to signify graft injury because steroid treatment results in improvement. Compared to palpation, echocardiography is more sensitive for assessing function of heterotopic pig heart xenografts. Echocardiography has, therefore, the potential to detect and treat early rejection episodes of heterotopic heart xenografts in nonhuman primates. This may help to achieve longer graft survival.

Characteristics of optic nerve damage induced by chronic intraocular hypertension in rat.

PURPOSE: To set up the Sharma's chronic intraocular hypertension model and investigate the intraocular pressure (IOP) as well as the optic nerve damage of this model in rat. METHODS: The operations of the chronic intraocular hypertension model were performed as described by Sharma in 60 Male Lewis albino rats. IOP was measured using the Tono-Pen XL immediately after surgery and then at 5 day, 2 week or 4 week intervals. Cresyl violet staining of whole-mounted retinas was used to label retinal ganglion cells (RGCs), then RGCs were counted. Paraphenylenediamine (PPD) staining was performed in the semi-thin cross sections of optic nerve of rat, in order to know whether the axons of optic nerve were degenerated or not. RESULTS: There were 47 rats with higher IOP after the episcleral veins cauterized in 60 rats. The ratio of elevated IOP was 78.3%. The IOPs were stable in 4 weeks. After cresyl violet staining, the RGCs loss was 11.0% and 11.3% was found in the central and peripheral retina respectively after 2 weeks of increased IOP. After 4 weeks of increased IOP, the loss of RGCs was 17% for the central retina and 24.6% for the peripheral retina. In the retinas without higher IOP, there was no loss of RGCs. PPD staining showed that optic nerve of rat with about 5.3% damage of axons located at the superior temporal region. Region of affected optic nerve 1 mm posterior to the globe by light microscope showed evidence of damaged axons with axonal swelling and myelin debris. CONCLUSION: Sharma's chronic intraocular hypertension model is a reproducible and effective glaucoma model, which mimics human glaucoma with chronically elevation IOP and induced RGCs loss and damage of optic nerve.

Gonadal hormones act extrinsic to the hippocampus to influence the density of hippocampal astroglial processes.

The important effects of estrogen on the morphology of hippocampal neurons are well established. The mechanisms leading to such changes, nevertheless, have proved confusingly complex, since interactions between glia and neurons, as well as neuronal influences from other brain fields, are involved. This study addresses the possibility that estrogen-sensitive projections from the medial septum/diagonal band of Broca induce astroglial reactions. Estrogen- and cholesterol-filled (controls) cannulae were implanted into the medial septum/diagonal band of Broca of adult ovariectomized rats. Comparative semiquantitative immunohistochemical analysis on the density of the glial fibrillary acidic protein-containing processes and cells were performed on hippocampal slices of locally estrogen-treated and control animals. Rats that received estrogen-filled cannulae showed a lower density of glial processes in the hippocampal CA1 and CA3 subfields than animals of the control group. These effects could not be observed in the dentate gyrus. Cell counts revealed no significant difference in the number of glial fibrillary acidic protein-positive cells in any of the examined areas. Two major conclusions can be drawn from these results. First, the data show that estrogen, in fact, has an indirect influence on hippocampal cells through septo-hippocampal projections. Furthermore, estradiol can have an indirect negative effect on hippocampal astrocytes, causing a reduction in the density of their processes.

Proliferation and apoptosis in the epithelium of the developing human cornea and conjunctiva.

To determine the distribution of proliferating and apoptotic cells in the human cornea during prenatal and early postnatal development, we examined sections of the bulbar conjunctiva, the limbus as well as the central and peripheral cornea between 11 weeks of gestation and 6 months after birth. The objective was to localize dividing cells by proliferating cell nuclear antigen-like immunoreactivity (PCNA-LI) and apoptotic cells by terminal transferase-mediated nick-end labeling (TUNEL). Before the 17th gestational week, PCNA-LI was absent in all 4 regions examined, indicating negligible cell proliferation during early development. After 20 weeks, strong PCNA-labeling was observed in all regions examined suggestive of high proliferative activity not only in the limbus and the bulbar conjunctiva, but also in the central and peripheral cornea. This rise in proliferative activity was followed by a steady decline: after 28 weeks, anti-PCNA staining gradually disappeared in the central and peripheral cornea, so that, at 6 months after birth, it was confined to the limbus and the bulbar conjunctiva, resembling the picture described for the adult cornea. TUNEL-positive cells were virtually absent in all 4 regions examined before the 38th gestational week. Apoptotic cells only started to appear at 38 weeks; at this stage, they were confined to the bulbar conjunctival epithelium. At 6 months after birth, TUNEL-positive cells were observed in the bulbar conjunctival epithelium and the entire cornea; the limbus, however remained devoid of apoptotic cells throughout the entire prenatal and early postnatal period. The present study for the first time localizes proliferating and apoptotic cells in the epithelium of the developing human cornea. Three stages of development can be distinguished: Minimal proliferation (until 17th week), vigorous proliferation over the entire cornea including the limbus and the bulbar conjunctiva (until 28th week) and gradual decrease in proliferative activity (after 28th week) accompanied by the appearance of apoptotic cells.

Reduced amplitude and delayed latency in foveal response of multifocal electroretinogram in early age related macular degeneration.

AIM: To explore the use of multifocal electroretinograms (MERG) in detecting early changes in age related macular degeneration (AMD). METHOD: 15 pre-AMD or early AMD eyes showing retinal drusen or irregular fundus pigmentation with window defects by fluorescein angiography (FA) and mildly decreased visual acuity were examined and compared with their asymptomatic fellow eyes. 20 age matched normal eyes were included as controls. MERG was recorded by a Veris system (version 3.0) using a 103 hexagon stimulus and 218 second total recording time per eye. The first order kernel was used to calculate amplitudes and latencies in three configurations: the nasal and the temporal areas, the superior and the inferior areas, and six concentric rings centred on the fovea. RESULTS: There were no significant differences in the amplitudes and the latencies between the different regions (nasal versus temporal and superior versus inferior) of the retina as well as between the different groups of eyes (normal, pre-AMD or early AMD, and the asymptomatic fellow eyes) in each region. Using the concentric configuration, the foveal amplitude of pre-AMD or early AMD eyes was significantly suppressed when compared with the age matched control group and their average latency was longer in the fovea than in outer rings and significantly prolonged when compared with the normal control group. Similar changes in amplitude and latency were also observed in the asymptomatic fellow eyes. CONCLUSION: Significant abnormality in the foveal amplitude and the foveal latency of MERG could be detected in pre-AMD or early AMD eyes as well as their asymptomatic contralateral eyes, suggesting MERG as a sensitive tool in detecting early foveal abnormalities in AMD.

Post-treatment at 12 or 18 hours with 3-aminobenzamide ameliorates retinal ischemia-reperfusion damage.

PURPOSE: The window of protection afforded by 3-aminobenzamide (3-ABA), a poly-(ADP-ribose) polymerase (PARP) inhibitor, against apoptotic loss of inner retinal elements after ischemia-reperfusion insult in rats was examined. METHODS: Ischemia-reperfusion injury to the retinas in albino Lewis rats was induced by elevated intraocular pressure (IOP) through cannulation of the anterior chamber with a needle connected to a saline column delivering a pressure of 110 mm Hg. The ischemic period was held at 60 minutes, and reperfusion was established immediately afterward. 3-Aminobenzamide (3-ABA) was administered intravitreally at 0, 4, 8, 12, 18, or 24 hours after reperfusion and its effect evaluated by morphology and morphometry of the inner retinas at 7 days after reperfusion. Immunohistochemistry of poly-(ADP-ribose), a product of PARP activity, and Western blot analysis for PARP were performed on retinas at 0, 4, 8, 12, 18, and 24 hours after reperfusion. RESULTS: Morphology and morphometry showed significantly better preserved inner retinas in animals receiving 3-ABA between 12 and 18 hours after reperfusion. Immunohistochemical study of poly-(ADP-ribose) showed elevated levels at the retinal ganglion cell layer and the inner nuclear layer at 12 and 18 hours after reperfusion. Western blot analysis of PARP showed a notable increase in the 116-kDa band (PARP) from 4 to 18 hours after reperfusion. CONCLUSIONS: Administration of 3-ABA at 12 or 18 hours after ischemia, when there was accumulation of poly-(ADP-ribose) in the inner retina, significantly ameliorated retinal ischemia-reperfusion injury. These findings, together with earlier reports from our laboratory, are consistent with a late and pivotal role of PARP in apoptotic loss of inner retinal elements after ischemia-reperfusion insult to the retina.

N -methyl- D -aspartate (NMDA) induced apoptosis in adult rabbit retinas.

Previously we showed that apoptosis is involved in N -methyl- D -aspartate (NMDA) induced excitotoxicity in adult rat retinas. Since rabbits have a higher endogenous level of glutamate in the retina and very different retinal structures, it is not clear if apoptosis is similarly involved in adult rabbit retinas after intravitreal injection of NMDA. In this study, we used ultrastructural features, TdT-mediated biotin-dUTP nick end labeling (TUNEL) and two caspase inhibitors to examine whether apoptosis is involved in NMDA-induced excitotoxicity in adult rabbit retinas. At 18 hr after an intravitreal injection of 400 nmoles NMDA, typical apoptotic features in degenerative cells in the retinal ganglion cell layer (RGCL) and the inner nuclear layer (INL) were noted by electron microscopy. TUNEL positive nuclei were detected in these layers as early as 4 hr showing maximal numbers at 18 hr. At 7 days, significant loss of nuclei from the RGCL was noted at the visual streak, the superior and the inferior retinas. These losses were abolished by simultaneous administration of MK-801 and ameliorated by YVAD, a caspase-1 inhibitor, but not by IETD, a caspase-8 inhibitor. These results indicated that, similar to adult rat retinas, apoptosis is involved in NMDA receptor-mediated excitotoxicity in rabbit retinas and that specific caspases may play important roles.

c-Fos protein in photoreceptor cell death after photic injury in rats.

PURPOSE: To examine the involvement of c-Fos protein in light-induced photoreceptor cell death in rats. METHODS: Thirty-two Lewis albino rats were exposed to green fluorescent light (480-520 nm) of 300 to 320 foot-candles (3228-3443.2 lux) for 3 hours, allowed to recover in the dark, and euthanatized at 0, 1, 3, 6, 12, 24, or 96 hours after light exposure. c-Fos was detected immunohistochemically and nicked DNA by in situ TdT-dUTP terminal nick-end labeling (TUNEL). Double labeling of c-Fos and DNA nicks was also performed. RESULTS: There was a time-dependent change in the number of c-Fos-positive photoreceptor nuclei after light injury, which paralleled the change in the number of TUNEL-positive nuclei. The temporal and spatial appearance of these nuclei also matched the appearance of pyknotic nuclei of the outer nuclear layer. Double-labeling study revealed that some c-Fos-positive nuclei were also TUNEL-positive nuclei. CONCLUSIONS: There was an acute accumulation of c-Fos protein in photoreceptors associated with cell death. This study further supports other studies showing that c-Fos is linked to apoptotic photoreceptor cell death.

N-methyl-D-aspartate (NMDA)--induced apoptosis in rat retina.

PURPOSE: The involvement of apoptosis in N-methyl-D-aspartate (NMDA)-induced excitotoxicity in adult rat retinas was examined. METHODS: Excitotoxic loss of inner retinal elements was induced by intravitreal injections of various concentrations of neutralized NMDA in adult albino Lewis rats. Tissue responses were quantified by measuring the inner retinal thickness (IRT) in plastic sections of the retinas and cell counts in the retinal ganglion cell layer in flatmount preparations of the whole retinas. Internucleosomal DNA fragmentation, a hallmark of apoptosis, was assayed with agarose DNA gel electrophoresis. The in situ TdT-mediated biotin-dUTP nick end labeling (TUNEL) method was used to locate nicked DNA in paraffin sections of the retinas. Ultrastructural changes of the degenerating cells were examined by electron microscopy. The efficacy of Ac-Tyr-Val-Ala-Asp-CMK (YVAD-CMK), a peptidyl caspase inhibitor, and 3-aminobenzamide (ABA), an inhibitor of poly(ADP-ribose) polymerase (PARP), in ameliorating the loss of inner retinal elements was evaluated using morphometry to examine the apoptotic pathways. RESULTS: Intravitreal injection of NMDA induced a dose-dependent loss of inner retinal elements as evidenced by the measurements of IRT and RGCCs. There were time- and dose-related appearances of internucleosomal fragmentation of retinal DNA and a time-related appearance of TUNEL-positive nuclei in the inner retinas after intravitreal NMDA injection. Ultrastructural features consistent with classic apoptotic changes were noted in degenerating cells in the retinal ganglion cell layer and the inner nuclear layer. Control retinas given vehicle, N-methyl-L-aspartate (the L-isomer of NMDA), or NMDA plus MK-801, a specific antagonist, did not show these changes. Simultaneous administration of NMDA and YVAD-CMK or ABA abolished or attenuated the loss of RGCCs in the posterior retinas. CONCLUSIONS: NMDA-induced excitotoxicity involved apoptosis and caspases and PARP may play important roles in the pathways.

Apoptosis and caspases after ischemia-reperfusion injury in rat retina.

PURPOSE: Extensive cell loss in the retinal ganglion cell layer (RGCL) and the inner nuclear layer (INL) was noted in a rat model of retinal ischemia-reperfusion injury by transient elevated intraocular pressure (IOP). The possible involvement of apoptosis and caspases was examined in this model of neuronal loss. METHODS: Transient elevated IOP was induced in albino Lewis rats through the insertion of a needle into the anterior chamber connected to a saline column. Elevated IOP at 110 mm Hg was maintained for 60 minutes. Groups of animals were euthanatized at various times after reperfusion, and their retinas were evaluated by morphology, agarose gel electrophoresis of DNA, in situ terminal deoxynucleotidyl transferase-mediated biotin-deoxyuridine triphosphate nick-end labeling (TUNEL), immunohistochemistry of caspases II (ICH1) and III (CPP32), and morphometry. YVAD.CMK, a tetrapeptide inhibitor of caspases, was used to examine the involvement of caspases. RESULTS: A marked ladder pattern in retinal DNA gel analysis, typical of internucleosomal DNA fragmentation and characteristic of apoptosis, was present 12 and 18 hours after reperfusion. Labeling of nuclei in the RGCL and the inner nuclear layer (INL) by TUNEL was noted between 8 and 18 hours after reperfusion. Histologic and ultrastructural features typical of apoptosis were also observed in the inner retina after ischemia. YVAD.CMK administered during the ischemic period inhibited apoptotic fragmentation of retinal DNA and ameliorated the tissue damage. When administered intravitreally 0, 2, or 4 hours after reperfusion, YVAD.CMK was also effective in preserving the inner retina but had no significant effect when administered 6 or 8 hours after reperfusion. The inner retina showed transient elevated immunoreactivity of caspases II and III 4 and 8 hours after reperfusion. CONCLUSIONS: Retinal ischemia-reperfusion after transient elevated IOP induced apoptosis of cells in the retinal ganglion cell layer and the INL. Caspases may have a pivotal role in the early events of the apoptotic pathway(s). Rescue by using anti-apoptotic agents after ischemia-reperfusion is feasible.