Multiplex PCR-based genotyping of Salmonella Enteritidis and Salmonella Typhimurium from food sources and assessment of their antimicrobial resistance profiles in Egypt.

BACKGROUND: Salmonellosis is a widespread zoonotic disease that poses a significant threat to livestock and public health. This study aimed to serotype 20 Salmonella isolates obtained from sixty retail chicken meats, assess Salmonella contamination from eggs, and evaluate antibiotic resistance profiles. METHODS AND RESULTS: Twenty eggs were randomly collected in the new Borg El Arab market. Bacterial isolation was carried out utilizing both traditional culture, biochemical, and PCR methods. Among the twenty eggs analyzed, three (15%) tested positive for Salmonella, while the remaining seventeen (85%) were confirmed as negative. Genotyping through multiplex PCR revealed the presence of two S. Enteritidis and other serovar, with the use of three specific gene sets: a random sequence for Salmonella spp., sdfI gene for S. Enteritidis, and flagellin (fliC gene) for S. Typhimurium. Out of the 20 isolates obtained from chicken meat, five (25%) were identified as S. Typhimurium, and three (15%) were classified as S. Enteritidis. All isolates sourced from chicken meat exhibited resistance to Rifampicin and Amoxicillin, with 90% displaying sensitivity to cefotaxime, gemifloxacin, and Erythromycin. Importantly, S. Blegdam, identified via serological methods, displayed resistance to all tested antibiotics. For the three isolates obtained from eggs, 66.6% showed sensitivity to cefotaxime, erythromycin, cefuraxime, and cefaclor, while displaying complete resistance (100%) to Amoxicillin, rifampicin, clarithromycin, and cefadroxil. Notably, one serovar exhibited absolute resistance to all tested drugs. CONCLUSION: Stakeholders must implement strict control measures and rationalize antibiotic use in veterinary and human medicine due to the rise of antibiotic-resistant strains.

Renal mass biopsy - a practical and clinicopathologically relevant approach to diagnosis.

Advancements in imaging modalities have increased the frequency of renal mass discovery. Imaging has typically been considered sufficient to guide management for a large proportion of these tumours, but renal mass biopsies (RMBs) have an increasing role in determining malignancy and can be a valuable tool for preventing unnecessary surgery in patients with benign tumours. A structured approach should be used to help to navigate the expanding repertoire of renal tumours, many of which are molecularly defined. In terms of tumour subtyping, the pathologist's strategy should focus on stratifying patients into clinically different prognostic groups according to our current knowledge of tumour behaviour, including benign, low-grade or indolent, intermediate malignant or highly aggressive. Crucial pathological features and morphological mimicry of tumours can alter the tumour's prognostic group. Thus, pathologists and urologists can use RMB to select patients with tumours at a reduced risk of progression, which can be safely managed with active surveillance within a tailored imaging schedule, versus tumours for which ablation or surgical intervention is indicated. RMB is also crucial in the oncological setting to distinguish between different high-grade tumours and guide tailored management strategies.

Influence of age and sex on physical, cardiac electrical and functional alterations in progressive hyperoxia treatment: A time course study in a murine model.

Oxygen supplementation is a widely used treatment for ICU patients. However, it can lead to hyperoxia, which in turn can result in oxidative stress, cardiac remodeling, and even mortality. This paper expands upon previous research conducted by our lab to establish time-dependent cardiac changes under hyperoxia. In this study, both young and aged mice (male and female) underwent 72 h of hyperoxia exposure and were monitored at 24-hour intervals for cardiac electrophysiological and functional parameters using ECG and electrocardiogram data. Our analysis showed that young male mice experienced significant weight loss as well as significant lung edema by 48 h. Although young male mice were highly susceptible to physical changes, they were resistant to early cardiac functional and electrophysiological changes compared to the other groups. Both young and aged female and aged males developed functional impairments by 24 h of hyperoxia exposure. Furthermore, sex and age differences were noted in the onset of electrophysiological changes. While some groups could resist early cardiac remodeling, our data suggests that 72 h of hyperoxia exposure is sufficient to induce significant cardiac remodeling across all age and sex groups. Our data establishes that time-dependent cardiac changes due to oxygen supplementation can have devastating consequences even with short exposure periods. These findings can aid in developing clinical practices for individuals admitted to the ICU by elucidating the impact of aging, sex, and length of stay under mechanical ventilation to limit hyperoxia-induced cardiac remodeling.

Influence of Age on Hyperoxia-Induced Cardiac Pathophysiology in Type 1 Diabetes Mellitus (T1DM) Mouse Model.

Mechanical ventilation often results in hyperoxia, a condition characterized by excess SpO2 levels (>96%). Hyperoxia results in changes in the physiological parameters, severe cardiac remodeling, arrhythmia development, and alteration of cardiac ion channels, all of which can point toward a gradual increase in the risk of developing cardiovascular disease (CVD). This study extends the analysis of our prior work in young Akita mice, which demonstrated that exposure to hyperoxia worsens cardiac outcomes in a type 1 diabetic murine model as compared to wild-type (WT) mice. Age is an independent risk factor, and when present with a major comorbidity, such as type 1 diabetes (T1D), it can further exacerbate cardiac outcomes. Thus, this research subjected aged T1D Akita mice to clinical hyperoxia and analyzed the cardiac outcomes. Overall, aged Akita mice (60 to 68 weeks) had preexisting cardiac challenges compared to young Akita mice. Aged mice were overweight, had an increased cardiac cross-sectional area, and showed prolonged QTc and JT intervals, which are proposed as major risk factors for CVD like intraventricular arrhythmias. Additionally, exposure to hyperoxia resulted in severe cardiac remodeling and a decrease in Kv 4.2 and KChIP2 cardiac potassium channels in these rodents. Based on sex-specific differences, aged male Akita mice had a higher risk of poor cardiac outcomes than aged females. Aged male Akita mice had prolonged RR, QTc, and JT intervals even at baseline normoxic exposure. Moreover, they were not protected against hyperoxic stress through adaptive cardiac hypertrophy, which, at least to some extent, is due to reduced cardiac androgen receptors. This study in aged Akita mice aims to draw attention to the clinically important yet understudied subject of the effect of hyperoxia on cardiac parameters in the presence of preexisting comorbidities. The findings would help revise the provision of care for older T1D patients admitted to ICUs.

Risk assessment supporting the establishment of a maximum residue limit for ractopamine in beef liver, applicable in the Arab Republic of Egypt.

In 2012, the Codex Alimentarius Commission adopted maximum residue limits (MRLs) for ractopamine in pig and cattle tissues. Egypt, a country that records a high consumption of beef liver, conducted a health risk assessment to estimate the risks associated with the adoption of Codex MRLs and the possible adoption of alternative values that may offer higher protection. Ractopamine was characterized based on previous assessments performed by international regulatory agencies, and an acceptable daily intake was set at 1 µg/kg bw for both chronic and acute ractopamine exposure. Beef liver consumption data for the Egyptian population were collected through a field survey (529 households, 1929 individuals). The standard body weight of 60 kg was used, as well as 70 kg, as a potentially more representative weight for the Egyptian population. Simulations showed that when the MRL for ractopamine in beef liver is set to 40 µg/kg (Codex MRL) or 20 µg/kg, the health-based guidance value of 1 µg/kg bw was not exceeded, as a result of chronic or acute exposure. An MRL of 20 µg/kg of ractopamine in beef liver was shown to provide optimum protection of Egyptian consumers, considering other potential sources of ractopamine intake and abnormally high consumption patterns, and was therefore recommended for adoption in Egypt. This study presents the inputs, model, and results of the probabilistic risk assessment that supported such recommendation. PRACTICAL APPLICATION: Residues of veterinary drugs, such as ractopamine, accumulate in animal tissues and may pose a risk to consumers. Establishing maximum residue limits (MRLs) will help importers by giving them the necessary visibility for commercial trade. It will also benefit Egyptian consumers, large consumers of beef liver, who will be better protected with a lower MRL than the internationally recommended one.

Impact of age and sex on hyperoxia-induced cardiovascular pathophysiology.

Hyperoxia is characterized by pronounced inflammatory responses, pulmonary cell apoptosis, and adverse cardiac remodeling due to an excess supply of oxygen. Hyperoxic episodes are frequent in mechanically ventilated patients and are associated with in-hospital mortality. This study extends the analysis of prior published research by our group as it investigates the influence of age in male and female rodents exposed to hyperoxic conditions. Age is an independent cardiovascular risk factor, often compounded by variables like obesity, diabetes, and a decline in sex hormones and their receptors. This study simulates clinical hyperoxia by subjecting rodents to > 90 % of oxygen for 72 h and compares the changes in cardiac structural and functional parameters with those exposed to normal air. While in both sexes conduction abnormalities with ageing were discernible, aged females owing to their inherent higher baseline QTc, were at a higher risk of developing arrhythmias as compared to age-matched males. Quantitative real-time RT-PCR and western blot analysis reflected altered expression of cardiac potassium channels, resulting in conduction abnormalities in aged female rodents. Unaffected by age and sex, hyperoxia-treated mice had altered body composition, as evidenced by a considerable reduction in body weight. Interestingly, compensatory hypertrophy observed as a protective mechanism in young males was absent in aged males, whereas protection of hearts from hyperoxia-induced cardiac hypertrophy was absent in aged female mice, both of which may be at least in part due to a reduction in sex steroid receptors and the systemic steroid levels. Finally, statistical analysis revealed that hyperoxia had the greatest impact on most of the cardiac parameters, followed by age and then sex. This data established an imperative finding that can change the provision of care for aged individuals admitted to ICU by elucidating the impact of intrinsic aging on hyperoxia-induced cardiac remodeling.

Study of Antibiotic Resistant Genes in Pseudomonas aeroginosa Isolated from Burns and Wounds.

Pseudomonas aeruginosa (P. aeruginosa) is frequently associated with infections with high mortality rates. The intrinsically high resistance to many antibiotics and multidrug resistance in the hospital setting is considered to be among the reasons for high pathogenicity of P. aeruginosa. In this study, a total of 200 wound and burn swabs were collected from patients. The collected specimens were examined for P. aeruginosa through biochemical and antibacterial sensitivity tests performed in the Microbiology Laboratory in College of Medicine, University of Kirkuk, Kirkuk, Iraq. The polymerase chain reaction was then used to detect mexA, mexB, mexR, and oprD genes. In total, 31 isolates of P. aeruginosa were collected from 200 patients with wounds and burns. Most cases were isolated from 23 (74.19%) and 8 (25.80%) wound and burn swabs, respectively. Antibiotic sensitivity was tested on all isolates against 17 antimicrobial agents. The obtained results revealed a high resistance rate to gentamicin, trimethoprim, amikacin, and amoxicillin, and a low resistance rate was observed to ceftazidime, tobramycin, levofloxacin, cotrimoxazole, ciprofloxacin, and aztreonam. Regarding antibiotic resistance, mexB, mexR, and oprD genes were observed in three isolates, in which mexB and mexR were detected in two isolates, and only one isolate carried mexA gene.

The implications of hyperoxia, type 1 diabetes and sex on cardiovascular physiology in mice.

Oxygen supplementation, although a cornerstone of emergency and cardiovascular medicine, often results in hyperoxia, a condition characterized by excessive tissue oxygen which results in adverse cardiac remodeling and subsequent injurious effects to physiological function. Cardiac remodeling is further influenced by various risk factors, including pre-existing conditions and sex. Thus, the purpose of this experiment was to investigate cardiac remodeling in Type I Diabetic (Akita) mice subjected to hyperoxic treatment. Overall, we demonstrated that Akita mice experience distinct challenges from wild type (WT) mice. Specifically, Akita males at both normoxia and hyperoxia showed significant decreases in body and heart weights, prolonged PR, QRS, and QTc intervals, and reduced %EF and %FS at normoxia compared to WT controls. Moreover, Akita males largely resemble female mice (both WT and Akita) with regards to the parameters studied. Finally, statistical analysis revealed hyperoxia to have the greatest influence on cardiac pathophysiology, followed by sex, and finally genotype. Taken together, our data suggest that Type I diabetic patients may have distinct cardiac pathophysiology under hyperoxia compared to uncomplicated patients, with males being at high risk. These findings can be used to enhance provision of care in ICU patients with Type I diabetes as a comorbid condition.

A case series of brain abscesses: an eleven-year retrospective single center study.

INTRODUCTION: We lack data on the epidemiology and management of brain abscesses in the Middle East. The aim of this study is to report a case series of brain abscesses admitted at a tertiary care center in Lebanon, between January 2008 and December 2018. METHODOLOGY: This retrospective study aimed at determining the demographic data, treatment, and correlations between different studied variables with prognosis of patients that received treatment. RESULTS: Forty-one patients (30 males) were included with a median age of 37 years (2-85). The analysis showed that the classic triad of fever, headache and neurologic deficit was only present in 12% of patients on admission. The source of infection was contiguous in 36.5%, post surgical in 32%, and distant in 17% of cases. Stereotactic biopsy was performed in 41.5% of patients, and craniotomy in 19.5%. A microorganism was isolated in 63% of patients (26 cases). The most used antibiotics were carbapenems (46%) and glycopeptides (66%). Eighty percent of patient (33) had a good outcome. A worse prognosis was significantly correlated with immunosuppression and multiple cerebral abscesses. CONCLUSIONS: Brain abscess remains a relatively rare condition.

Diagnostic and Prognostic Potential of Retinal Biomarkers in Early On-Set Alzheimer's Disease.

OBJECTIVE: Accumulating evidence suggests that the eye can be used in the assessment of early on-set Alzheimer's disease (AD). The eye offers a natural window to the brain through the retina. The retina and brain share common developmental origins and patho-physiological origins and mechanisms, having been sequestered from it during early development, but retaining its connections with the brain via the optic nerve. Therefore, it is well understood that neurological abnormalities have a direct profound impact on the retina. Recent studies suggest an array of physiological and pathological changes in the retina in dementia and specifically in AD. There are also reports on imaging the two hallmark proteins of the disease, extracellular amyloid beta peptides and intracellular hyper phosphorylated tau protein, as a proxy to neuroimaging. RESULTS: In this review, we summarise retinal structural, functional and vascular changes reported to be associated with AD. We also review techniques employed to image these two major hall mark proteins of AD and their relevance for early detection of AD.

Resveratrol as a Potential Therapeutic Candidate for the Treatment and Management of Alzheimer's Disease.

Resveratrol (3,4',5-trihydroxystilbene) is a naturally occurring phytochemical present in red wine, grapes, berries, chocolate and peanuts. Clinically, resveratrol has exhibited significant antioxidant, anti-inflammatory, anti-viral, and anti-cancer properties. Although resveratrol was first isolated in 1940, it was not until the last decade that it was recognised for its potential therapeutic role in reducing the risk of neurodegeneration, and Alzheimer's disease (AD) in particular. AD is the primary cause of progressive dementia. Resveratrol has demonstrated neuroprotective effects in several in vitro and in vivo models of AD. Apart from its potent antioxidant and anti-inflammatory roles, evidence suggests that resveratrol also facilitates non-amyloidogenic breakdown of the amyloid precursor protein (APP), and promotes removal of neurotoxic amyloid beta (Aß) peptides, a critical step in preventing and slowing down AD pathology. Resveratrol also reduces damage to neuronal cells via a variety of additional mechanisms, most notably is the activation of NAD(+)-dependent histone deacetylases enzymes, termed sirtuins. However in spite of the considerable advances in clarifying the mechanism of action of resveratrol, it is unlikely to be effective as monotherapy in AD due to its poor bioavailability, biotransformation, and requisite synergism with other dietary factors. This review summarizes the relevance of resveratrol in the pathophysiology of AD. It also highlights why resveratrol alone may not be an effective single therapy, and how resveratrol coupled to other compounds might yet prove an effective therapy with multiple targets.

Differential expression of sirtuins in the aging rat brain.

Although there are seven mammalian sirtuins (SIRT1-7), little is known about their expression in the aging brain. To characterize the change(s) in mRNA and protein expression of SIRT1-7 and their associated proteins in the brain of "physiologically" aged Wistar rats. We tested mRNA and protein expression levels of rat SIRT1-7, and the levels of associated proteins in the brain using RT-PCR and western blotting. Our data shows that SIRT1 expression increases with age, concurrently with increased acetylated p53 levels in all brain regions investigated. SIRT2 and FOXO3a protein levels increased only in the occipital lobe. SIRT3-5 expression declined significantly in the hippocampus and frontal lobe, associated with increases in superoxide and fatty acid oxidation levels, and acetylated CPS-1 protein expression, and a reduction in MnSOD level. While SIRT6 expression declines significantly with age acetylated H3K9 protein expression is increased throughout the brain. SIRT7 and Pol I protein expression increased in the frontal lobe. This study identifies previously unknown roles for sirtuins in regulating cellular homeostasis and healthy aging.

Accelerating Alzheimer's research through 'natural' animal models.

PURPOSE OF REVIEW: Alzheimer's disease is a complex multifactorial age-related neurodegenerative disorder. Current transgenic animal models do not fully recapitulate human Alzheimer's disease at the molecular, cellular and behavioural levels. This review aims to address the clinical relevance of using 'physiologically' aged rats, dogs and Octodon degus, as more representative 'natural' ecologically valid models to elucidate mechanistic aspects of Alzheimer's disease, and for the development of therapeutic agents to attenuate age-related cognitive decline. RECENT FINDINGS: Aged rats, dogs and O. degus decline cognitively and ultimately develop Alzheimer's disease-like symptoms in response to the natural ageing process. Aged rats provide a tractable and popular model to examine the neurobiological basis underlying cognitive decline with age, but they do not develop Alzheimer's disease pathology. Progressive accumulation of abnormal amyloid-beta in extracellular plaques and surrounding cerebral vasculature is a common feature in human Alzheimer's disease, aged canine model and most nonhuman primates. Interestingly, the O. degus develops amyloid-beta deposits, neurofibrillary tangles containing hyperphosphorylated tau protein, altered cholinergic transmission and cognitive deficits analogous to those observed in Alzheimer's disease. Natural animal models better represent the full pathophysiology of Alzheimer's disease and are not only a viable alternative to transgenic models, but also are arguably the preferable model. SUMMARY: 'Natural' models are useful to elucidate the neurobiological basis of Alzheimer's disease and develop effective therapeutic strategies that can be translated into human clinical trials.

Mapping NAD(+) metabolism in the brain of ageing Wistar rats: potential targets for influencing brain senescence.

Over the last decade, the importance of NAD(+) has expanded beyond its role as an essential cofactor for energy metabolism. NAD(+) has emerged as a major signalling molecule that serves as the sole substrate for several enzymatic reactions including the DNA repair enzyme, poly(ADP-ribose) polymerase (PARP), NAD-dependent protein deacetylases or CD38, and transcriptional factors by a new class of histone deacetylases known as sirtuins. NAD(+) levels are regulated by the metabolic status and cellular stress caused by oxidative stress and DNA damage. Since a detailed study of NAD(+) metabolism in the healthy ageing mammalian brain is nascent, we examined the effect of ageing on intracellular NAD(+) metabolism in different brain regions in female Wistar rats in young (3 months), middle aged (12 months) and older adults (24 months). Our results are the first to show a significant decline in intracellular NAD(+) levels and NAD:NADH ratio with ageing in the CNS, occurring in parallel to an increase in lipid peroxidation and protein oxidation (o- and m-tyrosine) and a decline in total antioxidant capacity. Hyperphosphorylation of H2AX levels was also observed together with increased PARP-1 and PARP-2 expression, and CD38 activity, concomitantly with reduced NAD(+) and ATP levels and SIRT1 function in the cortex, brainstem, hippocampus and cerebellum. Reduced activity of mitochondrial complex I-IV and impaired maximum mitochondrial respiration rate were also observed in the ageing rat brain. Among the multiple physiological pathways associated with NAD(+) catabolism, our discovery of CD38 as the major regulator of cellular NAD(+) levels in rat neurons indicates that CD38 is a promising therapeutic target for the treatment of age-related neurodegenerative diseases.

Connexin 30 expression and frequency of connexin heterogeneity in astrocyte gap junction plaques increase with age in the rat retina.

We investigated age-associated changes in retinal astrocyte connexins (Cx) by assaying Cx numbers, plaque sizes, protein expression levels and heterogeneity of gap junctions utilizing six-marker immunohistochemistry (IHC). We compared Wistar rat retinal wholemounts in animals aged 3 (young adult), 9 (middle-aged) and 22 months (aged). We determined that retinal astrocytes have gap junctions composed of Cx26, -30, -43 and -45. Cx30 was consistently elevated at 22 months compared to younger ages both when associated with parenchymal astrocytes and vascular-associated astrocytes. Not only was the absolute number of Cx30 plaques significantly higher (P<0.05) but the size of the plaques was significantly larger at 22 months compared to younger ages (p<0.05). With age, Cx26 increased significantly initially, but returned to basal levels; whereas Cx43 expression remained low and stable with age. Evidence that astrocytes alter connexin compositions of gap junctions was demonstrated by the significant increase in the number of Cx26/Cx45 gap junctions with age. We also found gap junctions comprised of 1, 2, 3 or 4 Cx proteins suggesting that retinal astrocytes use various connexin protein combinations in their gap junctions during development and aging. These data provides new insight into the dynamic and extensive Cx network utilized by retinal astrocytes for communication within both the parenchyma and vasculature for the maintenance of normal retinal physiology with age. This characterisation of the changes in astrocytic gap junctional communication with age in the CNS is crucial to the understanding of physiological aging and age-related neurodegenerative diseases.

Changes in kynurenine pathway metabolism in the brain, liver and kidney of aged female Wistar rats.

The kynurenine pathway of tryptophan catabolism plays an important role in several biological systems affected by aging. We quantified tryptophan and its metabolites kynurenine (KYN), kynurenine acid (KYNA), picolinic acid (PIC) and quinolinic acid (QUIN), and activity of the kynurenine pathway enzymes indoleamine 2,3-dioxygenase (IDO), tryptophan 2,3-dioxygenase (TDO) and quinolinic acid phosphoribosyltransferase (QPRTase), in the brain, liver and kidney of young, middle-aged and old female Wistar rats. Tryptophan levels and TDO activity decreased in all tissues with age. In contrast, brain IDO activity increased with age, while liver and kidney IDO activity decreased with age. The levels of KYN, KYNA, QUIN and PIC in brain all increased with age, while the levels of KYN in the liver and kidney showed a tendency to decrease. The levels of KYNA in the liver did not change, but the levels of KYNA in the kidney increased. The levels of PIC and QUIN increased significantly in the liver but showed a tendency to decrease in the kidney. QPRTase activity in both brain and liver decreased with age but was elevated in the kidney in middle-aged (12-month-old) rats. These age-associated changes in tryptophan metabolism have the potential to impact upon major biological processes, including lymphocyte function, pyridine (NAD(P)(H)) synthesis and N-methyl-d-aspartate (NMDA)-mediated synaptic transmission, and may therefore contribute to several degenerative changes of the elderly.

Age related changes in NAD+ metabolism oxidative stress and Sirt1 activity in wistar rats.

The cofactor nicotinamide adenine dinucleotide (NAD+) has emerged as a key regulator of metabolism, stress resistance and longevity. Apart from its role as an important redox carrier, NAD+ also serves as the sole substrate for NAD-dependent enzymes, including poly(ADP-ribose) polymerase (PARP), an important DNA nick sensor, and NAD-dependent histone deacetylases, Sirtuins which play an important role in a wide variety of processes, including senescence, apoptosis, differentiation, and aging. We examined the effect of aging on intracellular NAD+ metabolism in the whole heart, lung, liver and kidney of female wistar rats. Our results are the first to show a significant decline in intracellular NAD+ levels and NAD:NADH ratio in all organs by middle age (i.e.12 months) compared to young (i.e. 3 month old) rats. These changes in [NAD(H)] occurred in parallel with an increase in lipid peroxidation and protein carbonyls (o- and m- tyrosine) formation and decline in total antioxidant capacity in these organs. An age dependent increase in DNA damage (phosphorylated H2AX) was also observed in these same organs. Decreased Sirt1 activity and increased acetylated p53 were observed in organ tissues in parallel with the drop in NAD+ and moderate over-expression of Sirt1 protein. Reduced mitochondrial activity of complex I-IV was also observed in aging animals, impacting both redox status and ATP production. The strong positive correlation observed between DNA damage associated NAD+ depletion and Sirt1 activity suggests that adequate NAD+ concentrations may be an important longevity assurance factor.

Aging-related changes in astrocytes in the rat retina: imbalance between cell proliferation and cell death reduces astrocyte availability.

The aim of this study was to investigate changes in astrocyte density, morphology, proliferation and apoptosis occurring in the central nervous system during physiological aging. Astrocytes in retinal whole-mount preparations from Wistar rats aged 3 (young adult) to 25 months (aged) were investigated qualitatively and quantitatively following immunofluorohistochemistry. Glial fibrillary acidic protein (GFAP), S100 and Pax2 were used to identify astrocytes, and blood vessels were localized using Griffonia simplicifolia isolectin B4. Cell proliferation was assessed by bromodeoxyuridine incorporation and cell death by TUNEL-labelling and immunolocalization of the apoptosis markers active caspase 3 and endonuclease G. The density and total number of parenchymal astrocytes in the retina increased between 3 and 9 months of age but decreased markedly between 9 and 12 months. Proliferation of astrocytes was detected at 3 months but virtually ceased beyond that age, whereas the proportion of astrocytes that were TUNEL positive and relative expression of active caspase 3 and endonuclease G increased progressively with aging. In addition, in aged retinas astrocytes exhibited gliosis-like morphology and loss of Pax2 reactivity. A small population of Pax2(+)/GFAP(-) cells was detected in both young adult and aged retinas. The reduction in the availability of astrocytes in aged retinas and other aging-related changes reported here may have a significant impact on the ability of astrocytes to maintain homeostasis and support neuronal function in old age.

Perforin mediated apoptosis of cerebral microvascular endothelial cells during experimental cerebral malaria.

Cerebral malaria is a serious complication of Plasmodium falciparum infection. We have investigated the role of perforin in the pathogenesis of cerebral malaria in a murine model (Plasmodium berghei ANKA (PbA) infection). C57BL/6 mice demonstrated the typical neuropathological symptoms of experimental cerebral malaria infection from day 5p.i. and became moribund on day 6p.i. This pathology was not seen in PbA-infected, perforin-deficient (pfp-/-) mice. From days 5-6p.i. onwards there was a significant increase in mRNA for granzyme B and CD8, but not CD4, in brain tissue from PbA-infected C57BL/6 and pfp-/- mouse brains. Perforin mRNA was strongly increased in the brains of PbA-infected C57BL/6 mice on day 6p.i. Immunohistochemistry revealed increased perforin staining and elevated numbers of CD8(+) cells within the cerebral microvessels in PbA-infected C57BL/6 at days 5 and 6p.i. compared with uninfected animals. At day 6p.i., there were TUNEL-positive cells and activated caspase-3 positive cells of endothelial morphology in the CNS of PbA-infected C57BL/6 mice. The TUNEL-positive cells were greatly reduced in pfp-/- mice. These results suggest that CD8(+)T lymphocytes induce apoptosis of endothelial cells via a perforin-dependent process, contributing to the fatal pathogenic process in murine cerebral malaria.