A meta-analysis on the therapeutic efficacy of repetitive transcranial magnetic stimulation for cognitive functions in attention-deficit/hyperactivity disorders.

BACKGROUND: Therapeutic efficacies of repetitive transcranial magnetic stimulation (rTMS) for improving cognitive functions in patients with deficit/hyperactivity disorder (ADHD) remained unclear. The aim of this meta-analysis was to investigate the therapeutic efficacy of rTMS focusing on different cognitive performances. METHODS: Major databases were searched electronically from inception to February 2023 by using keywords mainly "rTMS" and "ADHD" to identify randomized controlled trials (RCTs) that investigated the therapeutic efficacy of rTMS for improving cognitive functions assessed by standardized tasks in patients with ADHD. The overall effect size (ES) was calculated as standardized mean difference (SMD) based on a random effects model. RESULTS: Meta-analysis of five RCTs with 189 participants (mean age of 32.78 and 8.53 years in adult and child/adolescent populations, respectively) demonstrated that rTMS was more effective for improving sustained attention in patients with ADHD compared with the control groups (SMD = 0.54, p = 0.001).Our secondary analysis also showed that rTMS was more effective for improving processing speed than the control groups (SMD = 0.59, p = 0.002) but not for enhancing memory or executive function. CONCLUSIONS: Our results supported the therapeutic efficacy of rTMS for improving sustained attention and processing speed. However, the limitation of available data warrants further studies to verify these findings.

Heat-induced seizures, premature mortality, and hyperactivity in a novel Scn1a nonsense model for Dravet syndrome.

Dravet syndrome (Dravet) is a severe congenital developmental genetic epilepsy caused by de novo mutations in the SCN1A gene. Nonsense mutations are found in ∼20% of the patients, and the R613X mutation was identified in multiple patients. Here we characterized the epileptic and non-epileptic phenotypes of a novel preclinical Dravet mouse model harboring the R613X nonsense Scn1a mutation. Scn1aWT/R613X mice, on a mixed C57BL/6J:129S1/SvImJ background, exhibited spontaneous seizures, susceptibility to heat-induced seizures, and premature mortality, recapitulating the core epileptic phenotypes of Dravet. In addition, these mice, available as an open-access model, demonstrated increased locomotor activity in the open-field test, modeling some non-epileptic Dravet-associated phenotypes. Conversely, Scn1aWT/R613X mice, on the pure 129S1/SvImJ background, had a normal life span and were easy to breed. Homozygous Scn1aR613X/R613X mice (pure 129S1/SvImJ background) died before P16. Our molecular analyses of hippocampal and cortical expression demonstrated that the premature stop codon induced by the R613X mutation reduced Scn1a mRNA and NaV1.1 protein levels to ∼50% in heterozygous Scn1aWT/R613X mice (on either genetic background), with marginal expression in homozygous Scn1aR613X/R613X mice. Together, we introduce a novel Dravet model carrying the R613X Scn1a nonsense mutation that can be used to study the molecular and neuronal basis of Dravet, as well as the development of new therapies associated with SCN1A nonsense mutations in Dravet.

Pilot study of a longitudinal integrated disaster and military medicine education program for undergraduate medical students.

Disaster medicine education in medical curricula is scarce and frequently nonexistent. It is reasonable to initiate educational approaches for physicians in this field at the medical school level. An understanding of disaster medicine and the health care system during massive casualty incidents has been recommended as an integral part of the medical curriculum in the United States and Germany.The goal of the reformed curriculum was to develop a longitudinal integrated disaster and military medicine education program extending from the first year to the sixth year based on previously separated clinical and military medicine topics. Emergency medicine physicians, military emergency medical technicians, and Tactical Combat Casualty Care instructors formed an interprofessional faculty group and designed a learning curriculum.A total of 230 medical students participated in the revised disaster preparedness curriculum. Satisfaction survey response rates were high (201/230, 87.4%). Most of the free-text comments on the program were highly appreciative. The students considered the number of teaching hours for the whole program to be adequate. The students showed significant improvements in knowledge and judgment regarding disaster medicine after the program.We found that medical students were highly interested, were appreciative of, and actively participated in this longitudinal integrated disaster and military medicine education program, but gaps existed between the students' scores and the educators' expectations. The educators believed that the students needed more disaster preparedness knowledge and skills.

The Ccr4-Not complex monitors the translating ribosome for codon optimality.

Control of messenger RNA (mRNA) decay rate is intimately connected to translation elongation, but the spatial coordination of these events is poorly understood. The Ccr4-Not complex initiates mRNA decay through deadenylation and activation of decapping. We used a combination of cryo-electron microscopy, ribosome profiling, and mRNA stability assays to examine the recruitment of Ccr4-Not to the ribosome via specific interaction of the Not5 subunit with the ribosomal E-site in Saccharomyces cerevisiae This interaction occurred when the ribosome lacked accommodated A-site transfer RNA, indicative of low codon optimality. Loss of the interaction resulted in the inability of the mRNA degradation machinery to sense codon optimality. Our findings elucidate a physical link between the Ccr4-Not complex and the ribosome and provide mechanistic insight into the coupling of decoding efficiency with mRNA stability.

Roles of the hypoximir microRNA-424/322 in acute hypoxia and hypoxia-induced pulmonary vascular leakage.

Acute mountain sickness (AMS) occurs in up to 25% of unacclimatized persons who ascend to 3000 m and can result in high-altitude pulmonary edema (HAPE). MicroRNAs (miRs) can regulate gene expression at the post-transcriptional level. Hypoxia selectively disrupts endothelial tight junction complexes through a hypoxia-inducible factor-1α (HIF-1α)-dependent mechanism. Though increased HIF-1α expression is associated with adaptation and protection from AMS development in the early stage of hypoxia, a downstream effector of HIF-1α, VEGF, can induce overzealous endothelial barrier dysfunction, increase vascular permeability, and ultimately result in HAPE and high-altitude cerebral edema. We hypothesized that the fine-tuning of downstream effectors by miRs is paramount for the preservation of endothelial barrier integrity and the prevention of vascular leakage. We found that several miRs were up-regulated in healthy volunteers who were subjected to a 3100-m height. By reviewing the literature and using online bioinformatics prediction software, we specifically selected miR-424 for further investigation because it can modulate both HIF-1α and VEGF. Hypoxia-induced miR-424 overexpression is HIF-1α dependent, and miR-424 stabilized HIF-1α, decreased VEGF expression, and promoted vascular endothelial cadherin phosphorylation. In addition, hypoxia resulted in endothelial barrier dysfunction with increased permeability; miR-424 thus attenuated hypoxia-induced endothelial cell senescence and apoptosis. miR-322 knockout mice were susceptible to hypoxia-induced pulmonary vascular leakage. miR-322 mimics improved hypoxia-induced pulmonary vascular leakage in vivo. We conclude that several miRs were up-regulated in healthy adult volunteers subjected to hypobaric hypoxemia. miR-424/322 could modulate the HIF-1α-VEGF axis and prevent hypoxia-induced pulmonary vascular leakage under hypoxic conditions.-Tsai, S.-H., Huang, P.-H., Tsai, H.-Y., Hsu, Y.-J., Chen, Y.-W., Wang, J.-C., Chen, Y.-H., Lin, S.-J. Roles of the hypoximir microRNA-424/322 in acute hypoxia and hypoxia-induced pulmonary vascular leakage.

mRNA Deadenylation Is Coupled to Translation Rates by the Differential Activities of Ccr4-Not Nucleases.

Translation and decay of eukaryotic mRNAs is controlled by shortening of the poly(A) tail and release of the poly(A)-binding protein Pab1/PABP. The Ccr4-Not complex contains two exonucleases-Ccr4 and Caf1/Pop2-that mediate mRNA deadenylation. Here, using a fully reconstituted biochemical system with proteins from the fission yeast Schizosaccharomyces pombe, we show that Pab1 interacts with Ccr4-Not, stimulates deadenylation, and differentiates the roles of the nuclease enzymes. Surprisingly, Pab1 release relies on Ccr4 activity. In agreement with this, in vivo experiments in budding yeast show that Ccr4 is a general deadenylase that acts on all mRNAs. In contrast, Caf1 only trims poly(A) not bound by Pab1. As a consequence, Caf1 is a specialized deadenylase required for the selective deadenylation of transcripts with lower rates of translation elongation and reduced Pab1 occupancy. These findings reveal a coupling between the rates of translation and deadenylation that is dependent on Pab1 and Ccr4-Not.

Kinect-based real-time audiovisual feedback device improves CPR quality of lower-body-weight rescuers.

BACKGROUND: Chest compression (CC) quality is associated with rescuer posture and body weight. We designed a Kinect module-based real-time audiovisual feedback (AVF) device to investigate the relationship between rescuer posture, body weight, and CC quality. METHODS: A total of 100 healthcare professionals were enrolled as participants in this randomized trial. A Kinect-based sensor system was used to monitor the depth and rate of CC and provide further real-time feedback. All participants were asked to perform continuous CC on a manikin with and without feedback for 2min individually in either a kneeling or standing position. RESULTS: A kneeling posture can provide higher rate of CC than a standing posture can (111.4±22.6 per minute vs. 99.1±18.9per minute, p value=0.005). Real-time AVF feedback can provide a better compression depth, rate, and effective compression ratio (6.16±1.88cm vs. 5.54±1.89cm, p value=0.02; 103.2±21.0/min vs. 96.7±25.8/min, p value=0.03; 62.6±28.0% vs. 51.0±33.2%, p value=0.004). Regardless of the effect of real-time feedback, the CC depth correlated to the rescuers' body weight. Rescuers who weighed below 71kg benefited from the Kinect module-based real-time AVF device in terms of improved CC quality. CONCLUSION: The Kinect-based AVF device can significantly improve CC quality in manikin training in rescuers with their body weight<71kg.

Short poly(A) tails are a conserved feature of highly expressed genes.

Poly(A) tails are important elements in mRNA translation and stability, although recent genome-wide studies have concluded that poly(A) tail length is generally not associated with translational efficiency in nonembryonic cells. To investigate whether poly(A) tail size might be coupled to gene expression in an intact organism, we used an adapted TAIL-seq protocol to measure poly(A) tails in Caenorhabditis elegans. Surprisingly, we found that well-expressed transcripts contain relatively short, well-defined tails. This attribute appears to be dependent on translational efficiency, as transcripts enriched for optimal codons and ribosome association had the shortest tail sizes, whereas noncoding RNAs retained long tails. Across eukaryotes, short tails were a feature of abundant and well-translated mRNAs. This seems to contradict the dogma that deadenylation induces translational inhibition and mRNA decay and suggests that well-expressed mRNAs accumulate with pruned tails that accommodate a minimal number of poly(A)-binding proteins, which may be ideal for protective and translational functions.

A Universal Code for mRNA Stability?

Precise elimination of maternal mRNAs plays a critical role during the maternal-to-zygotic transition (MZT) to promote developmental processing. Two new studies demonstrate that, in eukaryotes, codon-mediated decay is a conserved mechanism to shape maternal mRNA stability by affecting deadenylation rate in a translation-dependent manner. These studies add to a growing body of literature suggesting that translational elongation rates are a major determinant of mRNA stability.

The DEAD-Box Protein Dhh1p Couples mRNA Decay and Translation by Monitoring Codon Optimality.

A major determinant of mRNA half-life is the codon-dependent rate of translational elongation. How the processes of translational elongation and mRNA decay communicate is unclear. Here, we establish that the DEAD-box protein Dhh1p is a sensor of codon optimality that targets an mRNA for decay. First, we find mRNAs whose translation elongation rate is slowed by inclusion of non-optimal codons are specifically degraded in a Dhh1p-dependent manner. Biochemical experiments show Dhh1p is preferentially associated with mRNAs with suboptimal codon choice. We find these effects on mRNA decay are sensitive to the number of slow-moving ribosomes on an mRNA. Moreover, we find Dhh1p overexpression leads to the accumulation of ribosomes specifically on mRNAs (and even codons) of low codon optimality. Lastly, Dhh1p physically interacts with ribosomes in vivo. Together, these data argue that Dhh1p is a sensor for ribosome speed, targeting an mRNA for repression and subsequent decay.

An Elevated Glycemic Gap is Associated With Adverse Outcomes in Diabetic Patients With Community-Acquired Pneumonia.

Several studies argue against the association between admission hyperglycemia and adverse outcomes in infected diabetic patients. When investigating the association, it is necessary to consider preexisting hyperglycemia. The objective of this study was to assess whether stress-induced hyperglycemia, determined by the glycemic gap between admission glucose levels and A1c-derived average glucose levels adversely affects outcomes in diabetic patients admitted to hospital with community-acquired pneumonia (CAP).We retrospectively analyzed the glycemic gap and adverse outcomes of diabetic patients hospitalized because of CAP from June 1, 2007 to August 31, 2012 in single medical center in Taiwan.A total of 203 patients admitted with principal diagnosis of CAP and available data of glycemic gap.Patients with glycemic gaps ≥40 mg/dL had greater AUROC values for the development of adverse outcomes compared with acute hyperglycemia and long-term glycemic controls. Patients with an elevated glycemic gap had an odds ratio of 3.84 for the incidence of combined adverse outcomes. Incorporation of the glycemic gap into pneumonia severity index, CURB-65 or SMART-COP scores, increased the discriminative performance of predicting the development of adverse outcomes.Glycemic gaps were associated with adverse outcomes of diabetic CAP patients. The discriminative performance of the calculated glycemic gaps was comparable with those of current clinical scoring systems and may further increase the AUROC of each system.

The triterpenoids of Hibiscus syriacus induce apoptosis and inhibit cell migration in breast cancer cells.

BACKGROUND: Breast cancer-related mortality increases annually. The efficacy of current breast cancer treatments is limited, and they have numerous side effects and permit high recurrence. Patients with estrogen receptor (ER)-negative or triple-negative breast cancer are particularly difficult to treat. Treatment for this type of cancer is lacking, and its prognosis is poor, necessitating the search for alternative treatments. METHODS: This study screened Chinese herb Hibiscus syriacus extracts and identified a novel anti-cancer drug for patients with ER-negative breast cancer. The inhibitory effects on cell viability and migration were evaluated for each compound, and the molecular regulatory effects were evaluated on both mRNA and protein levels. RESULT: We found several triterpenoids including betulin (K02) and its derivatives (K03, K04, and K06) from H. syriacus inhibited human triple-negative breast cancer cell viability and migration but revealed smaller cytotoxic effects on normal mammalian epithelial cells. Betulin and its derivatives induced apoptosis by activating apoptosis-related genes. In addition, they activated p21 expression, which induced cell cycle arrest in breast cancer cells. Betulin (K02) and betulinic acid (K06) had stronger inhibitory effects on cell viability and migration than K03 and K04. CONCLUSIONS: H. syriacus extracts might inhibit breast cancer cell viability and induce apoptosis by activating p53 family regulated pathways and inhibiting AKT activation. H. syriacus extracts may provide important insight into the development of novel alternative therapies for breast cancer.

Codon optimality is a major determinant of mRNA stability.

mRNA degradation represents a critical regulated step in gene expression. Although the major pathways in turnover have been identified, accounting for disparate half-lives has been elusive. We show that codon optimality is one feature that contributes greatly to mRNA stability. Genome-wide RNA decay analysis revealed that stable mRNAs are enriched in codons designated optimal, whereas unstable mRNAs contain predominately non-optimal codons. Substitution of optimal codons with synonymous, non-optimal codons results in dramatic mRNA destabilization, whereas the converse substitution significantly increases stability. Further, we demonstrate that codon optimality impacts ribosome translocation, connecting the processes of translation elongation and decay through codon optimality. Finally, we show that optimal codon content accounts for the similar stabilities observed in mRNAs encoding proteins with coordinated physiological function. This work demonstrates that codon optimization exists as a mechanism to finely tune levels of mRNAs and, ultimately, proteins.

The physiological effects and quality of chest compressions during CPR at sea level and high altitude.

BACKGROUND: Rescuers that undergo acute ascent without acclimatization can experience acute mountain sickness. Although performing cardiopulmonary resuscitation (CPR) for a short period requires intensive effort at sea level, performing CPR at high altitude is even more exhausting and can endanger the rescuer. Therefore, we conducted a pilot study to compare the quality of resuscitation in health professionals at high altitude (3100 m) and that at sea level. METHODS: Thirty-eight participants were asked to performed continuous chest compression CPR (CCC-CPR) for 5 minutes at sea level and at high altitude. Cardiopulmonary resuscitation recording technology was used to objectively quantify the quality of the chest compressions (CCs), including the depth and rate thereof. RESULTS: At high altitude, rescuers showed a statistically significant decrease in blood oxygen saturation and an increase in systolic blood pressure, diastolic blood pressure, heart rate, and fatigue, as measured with the Borg score, after CCC-CPR compared with resting levels. The analysis of the time-dependent deterioration in the quality of CCC-CPR showed that the depth of CCs declined from the mean depth of the first 30 seconds after CCC-CPR to that at more than 120 seconds after CCC-CPR at both sea level and high altitude. The average number of effective CCs declined after CCC-CPR was performed for 1 minute at sea level and high altitude. CONCLUSIONS: The quality of CC rapidly declined at high altitude. At high altitude, the average number of effective CC decreases; and this decrease became significant after continuous CCs had been performed for 1 minute.

Amikacin-induced Fin Reduction is Mediated by Autophagy.

Despite its medical use, little is known about the mechanisms underlying amikacin-induced embryotoxicity, including fin reduction, in zebrafish. In this study, we examined the expression of well-known autophagy markers mTOR (target of rapamycin), atg10 (autophagy-related gene), atg12 and LC3 (mammalian homolog of Atg8) in amikacin-treated zebrafish embryos. Our results indicated that the mRNA expression level of atg12 in the amikacin-treated group was significantly increased by 1.5-fold (p<0.05) compared with the corresponding mock control group, while the expression levels of atg10 and mTOR were significantly decreased by 0.74-fold (p<0.05) and 0.58-fold (p<0.05), respectively. Western blot analysis revealed that LC3 protein expression was induced by amikacin. Taken together, these data suggest that amikacin-induced fin reduction is mediated by fin cell autophagy.

Toona sinensis Leaf Aqueous Extract Improves the Functions of Sperm and Testes via Regulating Testicular Proteins in Rats under Oxidative Stress.

Toona sinensis leaf (TSL) is commonly used as a vegetable and in spice in Asia. In this study, feeding with aqueous extract of TSL (TSL-A) alleviated oxidative stress and recovered the motility and functions of sperm in rats under oxidative stress. Protein expressions in testes identified by proteomic analysis and verified by Western blot demonstrated that TSL-A not only downregulated the level of glutathione transferase mu6 (antioxidant system), heat shock protein 90 kDa-ß (protein misfolding repairing system), cofilin 2 (spermatogenesis), and cyclophilin A (apoptosis) but also upregulated crease3-hydroxy-3-methylglutaryl-coenzyme A synthase 2 (steroidogenesis), heat shock glycoprotein 96, and pancreatic trypsin 1 (sperm-oocyte interaction). These results indicate that TSL-A promotes the functions of sperm and testes via regulating multiple testicular proteins in rats under oxidative stress, suggesting that TSL-A is a valuable functional food supplement to improve functions of sperm and testes for males under oxidative stress.

WNT10A plays an oncogenic role in renal cell carcinoma by activating WNT/ß-catenin pathway.

Renal cell carcinoma (RCC) is a malignancy with poor prognosis. WNT/ß-catenin signaling dysregulation, especially ß-catenin overactivation and WNT antagonist silencing, is associated with RCC carcinogenesis and progression. However, the role of WNT ligands in RCC has not yet been determined. We screened 19 WNT ligands from normal kidney and RCC cell lines and tissues and found that WNT10A was significantly increased in RCC cell lines and tissues as compared to that in normal controls. The clinical significance of increase in WNT10A was evaluated by performing an immunohistochemical association study in a 19-year follow-up cohort comprising 284 RCC and 267 benign renal disease (BRD) patients. The results of this study showed that WNT10A was dramatically upregulated in RCC tissues as compared to that in BRD tissues. This result suggests that WNT10A, nuclear ß-catenin, and nuclear cyclin D1 act as independent risk factors for RCC carcinogenesis and progression, with accumulative risk effects. Molecular validation of cell line models with gain- or loss-of-function designs showed that forced WNT10A expression induced RCC cell proliferation and aggressiveness, including higher chemoresistance, cell migration, invasiveness, and cell transformation, due to the activation of ß-catenin-dependent signaling. Conversely, WNT10A siRNA knockdown decreased cell proliferation and aggressiveness of RCC cells. In conclusion, we showed that WNT10A acts as an autocrine oncogene both in RCC carcinogenesis and progression by activating WNT/ß-catenin signaling.

Melatonin inhibits matrix metalloproteinase-9 (MMP-9) activation in the lipopolysaccharide (LPS)-stimulated RAW 264.7 and BV2 cells and a mouse model of meningitis.

We explored anti-inflammatory potential of melatonin against the lipopolysaccharide (LPS)-induced inflammation in vivo and in vitro. RAW 264.7 and BV2 cells were stimulated by LPS, followed by the treatment with melatonin or vehicle at various time intervals. In a mouse model of meningitis induced by LPS, melatonin (5mg/kg) or vehicle was intravenously injected at 30min postinsult. The activity of matrix metalloproteinase-2 (MMP-2) and metalloproteinase-9 (MMP-9) was determined by gelatin zymography. Nuclear factor-kappa B (NFκB) translocation and binding activity were determined by immunocytochemistry and electrophoretic mobility shift assay (EMSA). Our results showed that either pretreatment or cotreatment with melatonin at 50-500 µm effectively inhibited the LPS-induced proMMP-9 activation in the RAW 264.7 and BV2 cells, respectively (P<0.05). This melatonin-induced proMMP-9 inhibition remained effective when treatment was delayed up to 2 and 6hr postinsult for RAW 264.7 and BV2 cells, respectively (P<0.05 for both groups). Additionally, melatonin significantly attenuated the rises of circulatory and cerebral MMP-9 activity, respectively (P<0.05) and reduced the loss of body weight (P<0.05) in mice with meningitis. Moreover, melatonin (50µm) effectively inhibited nuclear factor-kappa B (NFκB) translocation and binding activity in the LPS-treated RAW 264.7 and BV2 cells, respectively (P<0.05). These results demonstrate direct inhibitory actions of melatonin against postinflammatory NFκB translocation and MMP-9 activation and highlight its ability to inhibit systemic and cerebral MMP-9 activation following brain inflammation.

Angiotensin converting enzyme DD genotype is associated with acute coronary syndrome severity and sudden cardiac death in Taiwan: a case-control emergency room study.

BACKGROUND: Angiotensin converting enzyme (ACE) gene insertion/deletion (I/D) polymorphisms have been associated with acute coronary syndrome (ACS); however, several controversial results have also been found in different studied populations. This hospital-based, emergency room, case-control study in Taiwan retrospectively investigated 111 ACS patients, and 195 non-coronary subjects as a control group, to study the effects of ACE I/D polymorphism in the most urgent ACS patients. ACE I/D polymorphisms were determined by polymerase chain reaction-based assays and their associations with ACS risk, severity, and sudden cardiac death were determined. RESULTS: The ACE DD genotype was associated with ACS incidence. The DD genotype was associated with a significant 4-fold higher risk of ACS in multivariate analysis (odds ratio (OR) = 4.295; 95% confidence interval (CI): 1.436-12.851, p = 0.009), and a 3.35-fold higher risk of acute myocardial infarction. DD genotype carriers also had more than 3-fold higher risks of stenosis in all the three coronary arteries, left anterior descending artery infarction, and anterior wall infarction. In addition, the DD genotype was also associated with a higher risk of sudden cardiac death (OR = 6.484, 95% CI: 1.036-40.598, p = 0.046). CONCLUSIONS: This study demonstrated that the ACE DD genotype is an independent risk factor for ACS, and in particular, for acute myocardial infarction. In addition, the ACE DD genotype is also associated with greater ACS severity and a higher risk of sudden cardiac death. ACE genotyping is recommended for patients with a history of ACS, and more intensive preventive care is suggested for patients with the DD genotype.

Optimal Percoll concentration facilitates flow cytometric analysis for annexin V/propidium iodine-stained ischemic brain tissues.

We sought to determine the optimal Percoll concentration for ischemic rat brain prepared for flow cytometric (FC) measurements. Animals were subjected to the right middle cerebral artery (MCA) occlusion, and were euthanized at 3, 12, 24, and 72 h after reperfusion onset. The brains were processed by different concentrations (unisolated, 20, 25, 30, or 40%) of Percoll and stained with annexin V/propidium iodine (PI). Ischemic brain damage was evaluated by FC analysis and image analysis for histologic sections. The relative susceptibility of different phenotypes of cells to necrotic and apoptotic damage were evaluated by the FC analyses for the immunohistochemistry, PI, and the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL)-processed brain tissues. Our results showed that FC analysis effectively detected the extent and maturation of apoptotic/necrotic brain damage, and the results were consistent with those determined from histologic brain sections. Neuron was more vulnerable to apoptosis than glia, whereas both cellular phenotypes were compatible in susceptibility for necrotic cell death. Percoll at a low concentration (20%) could effectively remove tissue debris without affecting membranous integrity of the injured neurons. Conversely, high percentages of Percoll (30-40%) substantially increased membranous damage for the injured cells. These results supported the application of FC to determine the extent and progression in time, as well as relative phenotypes of apoptotic/necrotic cell deaths following ischemic damage. We highlighted the use of Percoll at low percentages to facilitate the removal of tissue debris and to improve membrane integrity preservation for the injured neurons.