Genetic Variant XRCC1 rs1799782 (C194T) and Risk of Cancer Susceptibility in Indian Population: A Meta-analysis of Case–Control Studies

X-ray repair cross-complementing group 1 (XRCC1) plays a key role in the base excision repair pathway, as a scaffold protein that brings together proteins of the DNA repair complex. Several studies have reported contradictory results for XRCC1 exon 6 C>T (rs1799782) gene polymorphism and cancer risk in Indian population has provided inconsistent results. Therefore, we have performed this meta-analysis to evaluate the relationship between XRCC1 exon 6 C>T gene polymorphism and risk of cancer by published studies. We searched PubMed and Google scholar web databases to cover all studies published on association between XRCC1 exon 6 C>T gene polymorphism and cancer risk. The meta-analysis was carried out and pooled odds ratios (ORs) and 95% confidence intervals (95% CIs) were used to appraise the strength of association. In order to derive a more precise estimation of the association, A total of 3197 confirmed cancer cases and 3819 controls were included from eligible seventeen case-controls studies. Results from overall pooled analysis demonstrated suggested that that variant allele (T vs. C: OR 1.301, 95% CI 1.003–1.688, p = 0.047) was associated with the risk of overall cancer. Other genetic models; heterozygous (TC vs. CC: OR 1.108, 95% CI 0.827–1.485, p = 0.491), homozygous (TT vs. CC: OR 1.479, 95% CI 0.877–2.493, p = 0.142), dominant (TT+TC vs. CC: OR 1.228, 95% CI 0.899–1.677, p = 0.196) and recessive (TT vs. TC+CC: OR 1.436, 95% CI 0.970–2.125, p = 0.071) did not reveal statistical association. Publication bias observation was also considered and none was detected during the analysis. The present meta-analysis suggested that the variant allele T of XRCC1 exon 6 gene polymorphism was associated with the risk of cancer. It is therefore pertinent to confirm this finding in a large sample size to divulge the mechanism of this polymorphism and cancer risk in Indian population.     

Hyperinsulinemia and Hypoadiponectinemia are Associated with Increased Risk for Occurrence of Ovarian Cancer in Non-diabetic Women of North Indian Population

Ovarian cancer has been emerged as a most common and lethal gynecological malignancy in India. High serum insulin and low adiponectin have been associated with increased risk of ovarian cancer. But their role in development of ovarian cancer is conflicting and little evidence is available. We aimed to evaluate blood levels of insulin and adiponectin in epithelial ovarian cancer (EOC) patients and their association with the risk to develop EOC. The study included following three groups; Group 1: fifty cases of cytohistopathologically confirmed cases of EOC, Group 2: fifty age matched cases of benign ovarian conditions and Group 3: fifty ages matched healthy controls with no evidence of any benign or malignant ovarian pathology as ruled out by clinical examination and relevant investigations. Cytohistopathologically confirmed and newly diagnosed cases of EOC and benign ovarian cancer were included in this study. The median value of fasting serum insulin was significantly high (15.0 µlU/ml, P = 0.02) and adiponectin were significantly low (5.1 µg/ml, P < 0.001) in ovarian cancer patients compared to benign ovarian tumors and healthy controls group. A significant increase risk of ovarian cancer was found in high tertile (≥ 18.7 µlU/ml) of serum insulin level (OR = 2.7; 95% CI = 1.00–6.67, P = 0.04) and lower tertile (≤ 5.45 µg/ml) of adiponectin level (OR = 3.2; 95% CI = 1.10–9.71, P = 0.03). High serum insulin level and low adiponectin levels were significantly associated with increased risk for development of ovarian cancer.     

Distribution of Methionine Synthase Reductase (MTRR) Gene A66G Polymorphism in Indian Population

Methionine synthase reductase (MTRR) is an important enzyme of the folate/homocysteine pathway. It is responsible for regulation of methionine enzyme by reductive methylation. A common variant A66G is reported in the FMN-binding domain of the MTRR gene, which leads to substitution of isoleucine by methionine (I22M) in MTRR enzyme with reduced activity. Reduced catalytic activity of enzyme leads to high homocysteine concentration in blood and increases risk for numerous diseases. The frequency of A66G polymorphism varies in different ethnic groups. The present study has been designed to evaluate the frequency of MTRR A66G gene polymorphism in the Eastern UP population by PCR–RFLP method. Along with this we also performed a meta-analysis to evaluate the global prevalence of this polymorphism. Databases were screened to identified the eligible studies. The prevalence of the G allele and GG genotype was determined by the use of prevalence proportion with 95% CI. Open meta-analyst software was used for the meta-analysis. Total 1000 blood samples were analyzed, the frequencies of A and G alleles were 0.35 and 0.65 respectively. Meta-analysis results revealed that the prevalence of G allele and GG genotype were 49.4% (95% CI 40.6–58.1, p ≤ 0.001) and 24.3% (95% CI 17.8–30.9, p ≤ 0.001) respectively. In sub-group meta-analysis, the lowest frequency of G allele was found in South America (32.7%; 95% CI 14.1–51.3, p ≤ 0.001), and highest in Asia (56.4%; 95% CI 39.5–73.3, p ≤ 0.001). The results of the meta-analysis showed that the Asian population has the highest frequency of G allele and highest frequency of the GG genotype was found in the European population.     

Status of Vitamin D Receptor Gene Polymorphism and 25-Hydroxy Vitamin D Deficiency with Essential Hypertension

Essential hypertension (EH) is a multifactorial and complex disease with high rate of incidence and associated co-morbidities. Previous studies do not provide unanimous results for the risk of hypertension and association with Fok I genotype frequency and serum vitamin D levels. Hence, this study was undertaken to determine the status of Fok I vitamin D receptor (VDR) gene polymorphism along with vitamin D levels and blood pressure in patients with EH. Four hundred (200 controls and 200 cases of essential hypertension) participants from general Indian population were enrolled in this study. Peripheral blood samples were collected for genotyping Fok I-VDR gene polymorphism using PCR–RFLP method whereas 25-OH vitamin D levels in serum were quantified using high performance liquid chromatography (HPLC). Significantly reduced 25-OH vitamin D levels were observed in patients with EH (24.04 ± 8.62 vs 50.46 ± 15.46) compared to control subjects (p = 0.0001). Homozygous recessive genotype ‘ff’ frequency was increased by 8.06 fold (CI: 3.71–17.47, p = 0.0001) in patients with EH compared to dominant ‘FF’ genotype frequency. In conclusion, recessive ‘ff’ genotype frequency correlates with reduced serum vitamin D levels and results in significantly increased systolic and diastolic blood pressures leading to predisposition of EH.     

Synergetic Interaction of HLA-DRB1*07 Allele and TNF-Alpha − 863 C/A Single Nucleotide Polymorphism in the Susceptibility to Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is an inflammatory autoimmune disease which is characterized by dysregulation of various cytokines propagating the inflammatory processes that is responsible for tissue damage. Tumor necrosis factor alpha (TNF-α) is one of the most important immunoregulatory cytokines that has been implicated in the different autoimmune diseases including SLE. Two hundred and two patients with SLE and 318 controls were included in the study. The TNF-α gene promoter region (from − 250 to − 1000 base pairs) was analyzed by direct Sanger’s DNA sequencing method to find promoter variants associated with South Indian SLE patients. We have analyzed six TNF-α genetic polymorphisms including, − 863C/A (rs1800630), − 857C/T (rs1799724), − 806C/T (rs4248158), − 646G/A (rs4248160), − 572A/C (rs4248161) and − 308G/A (rs1800629) in both SLE patients and controls. We did not find association of TNF-α gene promoter SNPs with SLE patients. However, the − 863A (rs1800630) allele showed association with lupus nephritis phenotype in patients with SLE (OR: 1.62, 95%CI 1.04–2.53, P = 0.034). We found serum TNF-α level was significantly elevated in SLE cases as compared to control and found no association with any of the polymorphisms. The haplotype analysis revealed a significant protective association between the wild TNF-α alleles at positions − 863C, − 857C, − 806C, − 646G, − 572A and − 308G (CCCGAG) haplotype with lupus nephritis phenotype (OR 0.53, 95% CI 0.35–0.82, P = 0.004). Additionally, the TNF-α − 863 C/A (rs1800630) polymorphism and HLA-DRB1*07 haplotype showed significant differences between SLE patients and controls (OR 4.79, 95% CI 1.73–13.29, P = 0.0009). In conclusion, TNF-α − 863A allele (rs1800630) polymorphism is associated with increased risk of nephritis in South Indian SLE patients. We also found an interaction between HLA-DRB1*07 allele with TNF-α − 863 C/A promoter polymorphism giving supportive evidence for the tight linkage disequilibrium between TNF-α promoter SNPs and MHC class II DRB1 alleles.     

Relationship of Brain-Derived Neurotrophic Factor with Interleukin-23, Testosterone and Disease Severity in Schizophrenia

Hormonal imbalance, inflammation and alteration in synaptic plasticity are reported to play a crucial role in the pathogenesis of schizophrenia. The objective of the study was to assess the serum levels of brain derived neurotrophic factor (BDNF) and its association with interleukin-23 (IL-23), testosterone and disease severity in schizophrenia. 40 cases and 40 controls were included in the study. Serum levels of BDNF, IL-23 and testosterone were estimated in all the subjects. Disease severity was assessed using Positive and Negative Syndrome Scale (PANSS). The study was designed in Tertiary care hospital, South India. The results were compared between two groups using Mann–Whitney U test. Spearman Correlation analysis was used to assess the association between biochemical parameters and PANSS. Interleukin-23 and testosterone were significantly increased and BDNF was significantly reduced in schizophrenia cases when compared with controls. BDNF was negatively correlated with IL-23 (r = − 400, p = 0.011), positive symptom subscale (r = − 0.393, p = 0.012), general psychopathology score subscale (r = − 407, p = 0.009) and total symptom subscale (r = − 404, p = 0.010). There was no significant association of IL-23 and testosterone with disease severity in schizophrenia cases. BDNF was reduced in schizophrenia cases and negatively associated with interleukin-23 and disease severity scores.     

Significance of Vitamin D Binding Protein in Assessing Vitamin D Status Among Under-Five Children

Despite ample sunshine, 50–90% Indian children have Vitamin D deficiency (VDD). This enigma of widespread VDD needs exploration especially among under-fives as physiological variations in Vitamin D Binding Protein (VDBP) levels could be potential confounders in the interpretation of total 25-hydroxyvitamin D [25(OH)D]. However, there is scarce information about relevance of VDBP levels in under-five age group. We therefore, explored association of VDBP levels among 1–5 year old children with VDD. Serum levels of 25(OH)D, VDBP, calcium, parathyroid hormone (PTH) and alkaline phosphatase were estimated in 210 apparently healthy children in the age group of 1–5 years. VDD was defined as serum 25(OH)D levels < 20 ng/ml as per the IOM classification. VDBP levels were classified as low if levels were < 168 μg/ml as per the kit. The prevalence of VDD was 79.5% (n = 167) and VDBP levels were low in 48.6% (n = 102) of children. 25(OH)D levels correlated positively with VDBP (r = 0.298, p = 0.0001). A significant number of children (52.7%) with VDD had low VDBP (p = 0.015). and despite adequate sun exposure, 43% of children showed VDD and 56.6% had low VDPB levels. The low VDBP levels largely explain low 25OHD levels without necessarily implying VDD. It may add a new dimension for better understanding of widespread VDD among under-five children. It thus, points towards the need for redefining cut offs and complete evaluation of vitamin D status among under-fives including VDBP.     

Methylenetetrahydrofolate Reductase C677T Polymorphism and Recurrent Pregnancy Loss Risk in Asian Population: A Meta-analysis

The C677T polymorphism of the methylenetetrahydrofolate reductase (MTHFR) gene was implicated to be associated with thrombophilia due to its role in catalyzing the formation of 5-methylenetetrahydrofolate, a co-substrate for the conversion of homocysteine to methionine. Several case–control studies were investigated MTHFR C677T polymorphism as risk for recurrent pregnancy loss (RPL). These studies rendered contradictory results, some indicating that the polymorphism is associated with the risk of RPL whereas others concluded there is no association. To shed light on these inconclusive findings, a meta-analysis of all available studies published from Asian population relating the C677T polymorphism to the risk of RPL was conducted. The following electronic databases were searched without language restrictions: PubMed, Google Scholars, Elsevier and Springer Link up to December, 2015. Meta-analysis was performed using MetaAnalyst and Mix version 1.7. Meta-analysis results suggested that MTHFR C677T polymorphism contributed to the increased RPL risk in Asian population using all five genetic models (for T vs. C: OR 1.35, 95 % CI 1.09–1.68, p = 0.009; for TT + CT vs. CC: OR 1.44, 95 % CI 1.14–1.82, p = 0.006; for CT vs. CC: OR 1.39, 95 % CI 1.07–1.8, p = 0.01; for TT vs. CC: OR 1.79, 95 % CI 1.23.2.6, p = 0.007; for TT vs. CT + CC: OR 1.61, 95 % CI 1.02–2.56, p = 0.04). In conclusion, this meta-analysis demonstrates a strong association between the MTHFR C677T variant and RPL in Asian population and raising the importance of the use of folate in its treatment and prevention.     

CFTR Gene Mutations and Asthma in Indian Children: A Case–Control Study

Cystic Fibrosis Trans membrane conductance regulator (CFTR) gene is an asthma susceptibility gene. In the present study we investigated the possible association of CFTR gene mutations in Indian asthmatic children as compared to controls. The study included 250 asthmatics and 250 age and sex matched controls. Case to control ratio for sample size was 1:1. Genotyping was performed for 24 CFTR gene mutations by ARMS-PCR and PCR–RFLP method. Among 24 CFTR gene mutations, heterozygous allele of R553X mutation was found in 4 (1.6 %) asthmatic cases and 2 (0.8 %) controls. Value of FVC and FEV1/FVC ratio were significantly lower in heterozygous individuals (p value <0.05). No significant difference was observed in the genotype and allele frequency of R553X mutation (OR = 1.339, 95 % CI = 0.755–2.374, p value = 0.685). Furthermore, all wild type homozygous alleles were observed in remaining 23 CFTR gene mutations. Our data concludes that R553X mutation was not significantly associated in Indian asthmatic children.     

Folate Pathway Gene Methylenetetrahydrofolate Reductase C677T Polymorphism and Alzheimer Disease Risk in Asian Population

The association between methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism and susceptibility to Alzheimers disease (AD) was controversial in previous studies. The present meta-analysis was designed to investigate the association of MTHFR C677T polymorphism with AD. Nine studies were identified by search of PubMed, Google Scholar, Elsevier, Springer Link databases, up to January 2013. Odds ratios (ORs) with corresponding 95 % confidence interval (CI) were calculated using fixed effects model or random effects model. All statistical analysis was done by Mix version 1.7. MTHFR C677T polymorphism had a significant association with susceptibility to AD in all genetic models (for T vs C: OR 1.29, 95 % CI 1.15–1.44, p < 0.0001; for TT + CT vs CC: OR 1.38, 95 % CI 1.16–1.364, p = 0.0002; for TT vs CC: OR 1.60, 95 % CI 1.25–2.04, p = 0.0001; for CT vs CC: OR 1.28, 95 % CI 1.1–1.53, p < 0.008; for TT vs CT + CC: OR 1.37, 95 % CI 1.12–1.67, p = 0.002). Results from present meta-analysis supported that the MTHFR C677T polymorphism was associated with an increased risk of AD in Asian population.     

Genetic Association of rs2237572 Cyclin-Dependent Kinase 6 Gene with Breast Cancer in Iraq

This case–control study is aimed to evaluate serum concentration of Cyclin-Dependent Kinase 6 (CDK6) and the genetic association between rs2237572 CDK6 gene and breast cancer (BC) in Iraq. To attain this goal, 80 patients with BC as cases and 80 healthy individuals as controls were included. Further, BC patients were sorted according to the molecular classification into four subtypes of Luminal A, Luminal B, Her2/neu enriched and TPN. Serum concentration of CDK6 enzyme, allelic and genotypic frequencies of rs2237572 CDK6, and the occurrence of BC phenotype and its subtypes in the studied population were investigated. ELISA technique was used to perform the biochemical testing, while the molecular analysis was achieved by real-time PCR, high resolution melting analysis, conventional PCR, as well as sequencing analysis. The results revealed no significant difference in serum concentration of CDK6 enzyme between patients and healthy controls (p > 0.05). Also, no significant differences were shown between BC patients subtypes (p > 0.05). The rs2237572 CDK6 genotypes were associated with the BC and affirmed that allele C was inherited as a recessive risk factor. Moreover, a highly significant difference between patients’ subtypes in the genotypic frequency of rs2237572 (p < 0.01) was noted. Furthermore, the association of rs2237572 genotypes and CDK6 serum concentration in BC patients showed a considered significant difference between C/C and T/T, C/C and T/C and the CDK6 level (p < 0.05). Nevertheless, T/T and T/C did not show any significant difference with the CDK6 level. Hence, it was concluded that the rs2237572 of CDK6 gene is significantly correlated with BC.     

Genetic Polymorphism of Angiotensin II Type 1 Receptors and Their Effect on the Clinical Outcome of Captopril Treatment in Arab Iraqi Patients with Acute Coronary Syndrome (Mid Euphrates)

Genetic variation in the angiotensin II type 1 receptor (AT1R) has an important effect on the outcome of acute coronary syndrome (ACS) initiated treatment with captopril. This study aims to investigate the impact of genetic polymorphism of AT1R (rs5186 and rs275651) on the ACS outcome in Iraqi patients treated with captopril. A total of 250 Iraqi individuals with ACS were included in this case—control study and they were divided into two study groups; Study group 1 included 125 participants who were prescribed captopril, 25 mg twice daily and study group 2 included 125 participants who received no captopril as part of their ACS treatment (control study). The AT1R gene (rs5186) CC genotype was found to be associated with ST-elevation myocardial infarction (STEMI) (Odd’s ratio (O.R) = 1.2, P = 0.7), while AC was associated with Non-ST-elevation myocardial infarction (NSTEMI) and unstable angina (UA) (O.R = 1.2, P = 0.8). AC genotype is more prone to have Percutaneous coronary intervention (PCI) after ACS attack (O.R = 1.2, P = 0.6). CC genotype had a risk to get less improvement (O.R = 1.6, P = 0.5), so might require higher doses of captopril during acute coronary insult. The AT1R gene (rs275651) AA genotype was associated with UA (O.R = 1.3, P = 0.9). AA and AT genotypes were more prone to have PCI after ACS attack (O.R = 3.9 P = 0.2, O.R = 3.5, P = 0.3 respectively) and thus requiring higher doses of captopril. We conclude that the AT1R rs5186, rs275651 genetic polymorphisms might partially affect the clinical outcome of ACS patients treated with captopril and might have captopril resistance which requires higher doses.     

On-chip three-dimensional tumor spheroid formation and pump-less perfusion culture using gravity-driven cell aggregation and balanced droplet dispensing

This paper presents a spheroid chip in which three-dimensional (3D) tumor spheroids are not only formed by gravity-driven cell aggregation but also cultured at the perfusion rates controlled by balanced droplet dispensing without fluidic pumps. The previous spheroid chips require additional off-chip processes of spheroid formation and extraction as well as bulky components of fluidic pumps. However, the present spheroid chip, where autonomous medium droplet dispensers are integrated on a well array, achieves the on-chip 3D tumor spheroid formation and perfusion culture using simple structure without bulky fluidic pumps. In the experimental study, we demonstrated that the spheroid chip successfully forms 3D tumor spheroids in the wide diameter range of 220 μm–3.2 mm (uniformity > 90%) using H358, H23, and A549 non-small cell lung cancer cells. At the pump-less perfusion culture (Q = 0.1–0.3 μl/min) of spheroids, the number of H358 cells in the spheroid increased up to 50% from the static culture (Q = 0 μl/min) and the viability of the cultured cells also increased about 10%. Therefore, we experimentally verified that the perfusion environment created by the spheroid chip offers a favourable condition to the spheroids with high increase rate and viability. The present chip achieves on-chip 3D tumor spheroid formation and pump-less perfusion culture with simple structure, thereby exhibiting potential for use in integrated in-vivo-like cell culture systems.     

Genetic Variant Arg399Gln G&gt;A of XRCC1 DNA Repair Gene Enhanced Cancer Risk Among Indian Population: Evidence from Meta-analysis and Trial Sequence Analyses

The X-ray repair cross-complementation group 1 (XRCC1) gene plays an important role in base excision repair pathway. Several studies have reported contradictory results for XRCC1 exon 10 (Arg399Gln, G23990A, rs25487) gene polymorphism and cancer risk in Indian population, making it difficult to interpret them. Therefore, we have conducted a meta-analysis to evaluate the more precise association between XRCC1 exon 10 G>A gene polymorphism and risk of cancer by published studies. We searched PubMed (Medline) and Google scholar web databases to cover all studies published on association between XRCC1 exon 10 G>A gene polymorphism and cancer risk until August 2016. Pooled odds ratios (ORs) and 95% confidence intervals (95% CIs) were used to appraise the strength of association. Heterogeneity, publication bias and sensitivity analysis were also assessed. Twenty-five published studies had fulfilled the inclusion criteria comprising 4131 confirmed cancer cases and 5013 controls. When all studies were polled together, overall significant association was found between XRCC1 exon 10 G>A polymorphism and cancer risk in variant allele carrier (A vs. G: OR 1.217, 95% CI 1.056–1.402, p = 0.007), homozygous (AA vs. GG: OR 1.359, 95% CI 1.036–1.783, p = 0.027), dominant (AA+AG vs. GG OR 1.208, 95% CI 1.006–1.450, p = 0.043) and recessive (AA vs. AG+GG: OR 1.315, 95% CI 1.029–1.680, p = 0.029) genetic models. Further sensitivity analysis supported the stability of our result by showing similar ORs before and after removal of a single study. The present meta-analysis suggested that the XRCC1 exon 10 G>A polymorphism contribute cancer risk in Indian population, and supports that individuals with risk allele A and AA genotype are at higher risk of developing cancer.     

Finite element analysis of helical flows in human aortic arch: A novel index

This study investigates the helical secondary flows in the aortic arch using finite element analysis. The relationship between helical flow and the configuration of the aorta in patients of whose three-dimensional images constructed from computed tomography scans was examined. A finite element model of the pressurized root, arch, and supra-aortic vessels was developed to simulate the pattern of helical secondary flows. Calculations indicate that most of the helical secondary flow was formed in the ascending aorta. Angle α between the zero reference point and the aortic ostium (correlation coefficient (r) = −0.851, P = 0.001), the dispersion index of the cross section of the ascending (r = 0.683, P = 0.021) and descending aorta (r = 0.732, P = 0.010), all correlated closely with the presence of helical flow (P < 0.05). Stepwise multiple linear regression analysis confirmed angel α to be independently associated with the helical flow pattern in therein (standardized coefficients = −0.721, P = 0.023). The presence of helical fluid motion based on the atherosclerotic risks of patients, including those associated with diabetes, hypertension, hyperlipidemia, or renal insufficiency, was also evaluated. Numerical simulation of the flow patterns in aortas incorporating the atherosclerotic risks may better explain the mechanism of formation of helical flows and provide insight into causative factors that underlie them.     

Microwells support high-resolution time-lapse imaging and development of preimplanted mouse embryos

A vital aspect affecting the success rate of in vitro fertilization is the culture environment of the embryo. However, what is not yet comprehensively understood is the affect the biochemical, physical, and genetic requirements have over the dynamic development of human or mouse preimplantation embryos. The conventional microdrop technique often cultures embryos in groups, which limits the investigation of the microenvironment of embryos. We report an open microwell platform, which enables micropipette manipulation and culture of embryos in defined sub-microliter volumes without valves. The fluidic environment of each microwell is secluded from others by layering oil on top, allowing for non-invasive, high-resolution time-lapse microscopy, and data collection from each individual embryo without confounding factors. We have successfully cultured mouse embryos from the two-cell stage to completely hatched blastocysts inside microwells with an 89% success rate (n = 64), which is comparable to the success rate of the contemporary practice. Development timings of mouse embryos that developed into blastocysts are statistically different to those of embryos that failed to form blastocysts (p–value < 10⁻¹⁰, two-tailed Student''s t-test) and are robust indicators of the competence of the embryo to form a blastocyst in vitro with 94% sensitivity and 100% specificity. Embryos at the cleavage- or blastocyst-stage following the normal development timings were selected and transferred to the uteri of surrogate female mice. Fifteen of twenty-two (68%) blastocysts and four of ten (40%) embryos successfully developed into normal baby mice following embryo transfer. This microwell platform, which supports the development of preimplanted embryos and is low-cost, easy to fabricate and operate, we believe, opens opportunities for a wide range of applications in reproductive medicine and cell biology.     

Microfluidic culture platform for studying neuronal response to mild to very mild axonal stretch injury

A new model for studying localised axonal stretch injury is presented, using a microfluidic device to selectively culture axons on a thin, flexible poly (dimethylsiloxane) membrane which can be deflected upward to stretch the axons. A very mild (0.5% strain) or mild stretch injury (5% strain) was applied to primary cortical neurons after 7 days growth in vitro. The extent of distal degeneration was quantified using the degenerative index (DI, the ratio of fragmented axon area to total axon area) of axons fixed at 24 h and 72 h post injury (PI), and immunolabelled for the axon specific, microtubule associated protein-tau. At 24 h PI following very mild injuries (0.5%), the majority of the axons remained intact and healthy with no significant difference in DI when compared to the control, but at 72 h PI, the DI increased significantly (DI = 0.11 ± 0.03). Remarkably, dendritic beading in the somal compartment was observed at 24 h PI, indicative of dying back degeneration. When the injury level was increased (5% stretch, mild injury), microtubule fragmentation along the injured axons was observed, with a significant increase in DI at 24 h PI (DI = 0.17 ± 0.02) and 72 h PI (DI = 0.18 ± 0.01), relative to uninjured axons. The responses observed for both mild and very mild injuries are similar to those observed in the in vivo models of traumatic brain injury, suggesting that this model can be used to study neuronal trauma and will provide new insights into the cellular and molecular alterations characterizing the neuronal response to discrete axonal injury.