Triticum aestivum L.) grown under two nitrogen regimes and in three sowing densities during four early plant growth stages (Zadok’s stages 14–32) in 2017. Imaging-based canopy cover was tested in correlation analysis for estimating dry weight, nitrogen uptake and nitrogen content. An active Greenseeker sensor and various established and newly developed vegetation indices and spectral unmixing from a passive hyperspectral spectrometer were used as alternative approaches and additionally tested for retrieving canopy cover. Before tillering (until Zadok’s stage 20), correlation coefficients for dry weight and nitrogen uptake with canopy cover strongly exceeded all other methods and remained on higher levels (R² > 0.60***) than from the Greenseeker measurements until tillering. From early tillering on, red edge based indices such as the NDRE and a newly extracted normalized difference index  were identified as best spectral methods for both traits whereas the Greenseeker and spectral unmixing correlated best with canopy cover. RGB-segmentation could be used as simple low-cost approach for very early growth stages until early tillering whereas the application of multispectral sensors should consider red edge bands for subsequent stages.Plant vigor is an important trait of field crops at early growth stages, influencing weed suppression, nutrient and water use efficiency and plant growth. High-throughput techniques for its evaluation are required and are promising for nutrient management in early growth stages and for detecting promising breeding material in plant phenotyping. However, spectral sensing for assessing early plant vigor in crops is limited by the strong soil background reflection. Digital imaging may provide a low-cost, easy-to-use alternative. Therefore, image segmentation for retrieving canopy cover was applied in a trial with three cultivars of winter wheat ( Increasing the efficiency in using fertilizer and pesticides in cropping systems has become a crucial challenge in current crop production. In recent years, spectral sensing has become a versatile tool for evaluating crop stands and determining fertilizer demand ,2. SensoCanopy sensing is not recommended in early growth stages, due to distortion by soil background scattering in sparse canopies ,10, whicA large number of vegetation indices has been developed over time to predict vegetation traits based on different spectral parameters, but their usage is sensor-dependent and has rarely been evaluated at early growth stages ,13. FreqBrightness and surface structure of the soil impact spectral measurements in sparse canopies ,24,25, iAlternatively, the total spectral information can be included through spectral unmixing, which represents the spectral signature as mixed information of two or more surface types, which are referred to as endmembers . ExampleEarly plant development is an important trait influencing early N-uptake, potential weed suppression and water use efficiency, all of which can be related to final yield potential. Detecting such traits is equally relevant in precision farming for potentially guiding early fertilization as well as for phenotyping for identifying superior genotypes ,36,37,38In remote sensing, image-based techniques involving only few wavebands have been prevalent for a long time . HoweverVarious attempts have been undertaken to use simple RGB-based pixel analysis to retrieve fractional canopy cover showing acceptable correlations up to r = 0.87 with aboveground biomass ,44. CompWhile biomass, nutrition or water status are key target traits in plant nutrition, digital images could also be used to quantify the amount or type of weed, to detect plant diseases and to estimate soil evaporation ,49,50,51Image-based techniques could hold multiple advantages such as lower sensor costs and robustness when consumer-grade cameras can be used . FurtherThe objective of this study was to evaluate the performance of a consumer-grade camera for detecting biomass, N-uptake and N-content at four early plant growth stages (Zadok’s stages 12–32), corresponding to leaf development until early stem elongation, in comparison to a simple active Greenseeker sensor and vegetation indices of a passive hyperspectral spectrometer in winter wheat. It is hypothesized that digital images will show better results in the early stages, while spectral remote sensing will be negatively influenced by soil backscattering. With increasing canopy cover (CC) and plant growth, spectral sensing could improve and replace digital-image analysis due to saturation of the green-pixels in the imaging approach.−1 and N2 receiving 60 kg N ha−1 for the first nitrogen dressing. The line winter wheat cultivars Anapolis, and Mulan and the hybrid wheat cultivar Hybery were grown on the sub-plot. Anapolis was distinguished by pronounced planophile growth until tillering. The second block was divided into three sowing rates including 150, 250 and 350 kernels m−² and the cultivars Anapolis and Hybery on the sub-plot. The field was comprised mostly of a homogeneous Cambisol with a silty clay loam soil textural class. The average annual precipitation in this region is approximately 800 mm with an average annual temperature of 8 °C. The previous crop was grass-clover.The experiment was established as a split-split plot design with three replicates at the Dürnast research station of the Technical University of Munich located in South-East Germany  and consisted of two parts: The first block was sub-divided into two main blocks with different N fertilizer application rates with N1 receiving 30 kg N haPlant sampling and sensor measurements were conducted at four dates during the early growing season in 2017 at the beginning of vegetation from March 15 to May 3, corresponding to Zadok’s growth stages 12–32 . Plant s® Handheld Crop Sensor  for retrieving NDVI, and from the bidirectional hyperspectral passive spectrometer Handyspec . The Handyspec yielded spectral readings from 300 nm to 1140 nm at a nominal resolution of 3.3 nm. It is equipped with two silicon diode array spectrometers  which measure simultaneously downwelling and upwelling radiation. It was calibrated prior to the measurements using a polytetrafluoroethylene (PTFE) white standard .Afterwards, spectral measurements were taken using the GreenSeekerThe actively measuring Greenseeker sensor emits light by two diodes and detects the reflection in the visible range around 656 nm and in the NIR range at 774 nm, with a band width of ~25 nm in both bands. The readings are directly provided as NDVI value. Both sensors were activated for approximately 5 s and delivered averaged sensor readings.Both sensors were held in nadir direction over the center of the sampling area. Hold at 60–70 cm above the canopy, the oval field of view of the Greenseeker stretched in parallel to the sowing direction has a length of 25–30 cm. For the Handyspec, the aperture angle of 25° resulted in a circular field of view giving a diameter of 25–30 cm at the same measurement height.Thereafter, total aboveground biomass of the marked sampling area was manually sampled and washed for removing adherent soil particles. Plant samples were dried for two days at 50 °C and weighed for the determination of the dry weight (DW) and were finely ground. Biomass N content (NC) was determined by NIR-spectroscopy . N-uptake (Nup) was calculated by multiplying DW with NC.Statistical analysis was conducted in the open source software R 3.4.2 . A two-wRGB images were subjected to color-based image segmentation using the open source software ImageJ , similarR. The estimated canopy coverage was calculated as ratio of the regression coefficient for dense vegetation to the sum of the regression coefficients of both endmembers.From the Greenseeker sensor, only one default NDVI-index was available. The hyperspectral Handyspec data was exploited in different ways. Several preselected narrow-band vegetation indices were calculated using the passive spectral data : The staThe image-based CC, the Greenseeker NDVI, all pre-defined, established spectral indices and the spectral unmixing estimate for canopy coverage obtained from the Handyspec measurements were used for testing simple linear correlations with the target traits DW, Nup and NC for each sampling date. Likewise, considering CC as target trait, the Greenseeker and Handyspec readings were tested for correlations with CC. Since the direction of the relationship between sensor methods and the target traits is often not of interest, the squared correlation coefficient (R²) was reported for more direct comparison of different methods.Mean and coefficient of variation (CV) of the R²-values from the tested sensor methods were retrieved over the four measurement dates for evaluating the level and stability of the models.In addition to established spectral vegetation indices, pairwise normalized difference indices were calculated from all possible band combinations and also tested in correlation analysis for each date*trait combination using the hsdar-package in R . The speIn the sowing density block, no significant cultivar effects were observed for the four target traits for any of the measurement dates (D) (not shown). Differences between sowing densities were observed on all days for DW and CC, for Nup on all days except on D4 but only on D4 for NC. In the nitrogen fertilization block, no fertilization effect was found. For cultivar effects, only Mulan showed significantly lower CC than Hybery and Anapolis on D3 and D4.Still, substantial differences between experimental plots were found. All traits increased exponentially over the measurement dates with the exception of NC . DW and −1 and 3.7 to 78 kg ha−1, respectively. DW and Nup were always closely correlated (R² = 0.91–0.99) whereas NC showed medium negative correlations with DW on D3 and D4 and with Nup on D4, indicating a strongly dominating effect of DW on Nup , when on average only 4.5% of the ground was covered by the canopy, until early stem elongation , the Greenseeker-NDVI as well as selected vegetation indices and spectral unmixing from the hyperspectral data, with the reference traits DW, NC and Nup for all dates . In addiNo significant correlations with N content (NC) were found using any of the tested established methods, both for D1 and D2. Correlations strongly increased for most other sensor method/rait combinations on D2. Again, NDRE yielded best correlations with DW and Nup  but was slightly outperformed by the spectral unmixing method for CC (R² = 0.56). Similar to D1, differences in R² values of NDRE and ATSAVI were small but EPVI performed less well (R² = 0.69 for Nup). However, models from WBI, PRI and REIP provided again lower correlations compared to the best indices. This was now also the case for the Greenseeker sensor (R² = 0.54 for Nup) in spite of still increasing R² for DW and Nup but decreasing R²-values for CC in comparison to D1. In contrast to all hyperspectral indices, CC used as sensor method followed an opposite trend and reached lower correlations than on D1, both with DW and Nup (0.65 and 0.67).Contrasting to D1 to D2, the overall index ranking changed and was not similar for all target values on D3, even if the level of correlations remained comparable. REIP, which strongly improved from D2, yielded now best R²-values with DW (0.77) and was slightly outperformed for NC by MTCI (0.35). However, these indices ranked not among the best indices for CC. The indices Greenseeker and PRI reached best correlations for CC (R² = 0.73), followed by ATSAVI and EPVI.On D4, correlations decreased for almost all sensor methods for DW and Nup compared to D3, with PRI, EPVI, ATSAVI, NDVI, MCARI and spectral unmixing showing the strongest reduction. While on D3, NDVI, NDRE, WBI, and CC formed a rather homogenous group with similar R² values for DW and Nup, NDVI yielded now weaker correlations. Still, REIP reached best correlations both with DW and Nup . However, for estimating CC, spectral unmixing and Greenseeker (both 0.70) reached the best correlations on this day. While the ranking of sensor methods was close between Nup and DW for all days (R² between correlations for both traits > 0.93), the index ranking substantially differed between CC and DW for D3 and D4.The best hyperspectral normalized difference indices found in the contour maps revealed a higher potential for the estimation of all traits compared to established hyperspectral vegetation indices. However, the relative increase in R² was stronger for measurement days and traits with lower correlation level from established sensor methods . Similar to the pre-defined vegetation indices, contour maps averaged over four measurement days for DW and Nup showed similar correlation patterns , Table 3For DW and Nup, a broad field of combinations of VIS/NIR bands yielded on average enhanced correlations (R² > 0.5), which however were exceeded by red edge/NIR-combinations . The patFor estimating CC, the pattern was similar as for DW, but without peaks in the red edge/NIR range. In contrast to the other traits, a second peak was visible for combinations in the red/red and red/green range (500–680 nm), with the maximum R² (0.55) reached by a narrow-banded spectral combination at 584 nm; 582 nm.The stability of index correlations from the contour maps analysis over the four measurement dates differed between spectral regions and was generally inverse to the mean R² . For DW The best vegetation indices for DW and Nup only comprised NIR/red edge combinations (purple) and a few NIR/NIR combinations (blue) followed by the VIS/NIR group (green) and combinations of two red edge bands (red), whereas VIS/red edge (black) and VIS/VIS (orange)combinations dropped in mean R². Compared to NIR/NIR and VIS/VIS indices, all combinations including red edge bands differed substantially less among each other in mean R².Unlike for DW and Nup, for CC, overall best correlations paired with lowest CV were found from VIS/NIR and VIS/VIS combinations. However, in contrast to DW and Nup, a clear narrowing at the bottom of the figure is missing.The mean R² values as depicted in Mean R² values of pre-defined and best contour map indices as listed in As for all contour map indices , a negatThe main objective of this study was to evaluate the performance of a consumer-grade camera for detecting biomass and N-uptake at four early growth stages (Zadok’s stages 12–32) in comparison to an active Greenseeker sensor and different vegetation indices of a passive hyperspectral spectrometer in winter wheat.Apart from the sowing density trial, mostly no significant treatment effects were found during early plant development. The sampling period was characterized by dry weather with little precipitation within three weeks following fertilization and cold temperature when higher amounts of rain fell from March 26 onwards. Thus, plant growth and the effect of N fertilization was limited whereas differentiation on plot level was also driven by heterogeneous plant development in the trials.From all sensor methods, largely similar correlations were found for DW compared to Nup, what can be attributed to the close correlation between both target traits on all days, whereas little variation in Nup was due to differences in NC. However, as reported for later stages as dilution effects ,63, an iThe correlations of image-based canopy cover (CC) with DW and Nup decreased over time. With the plots approaching canopy coverage on D4, CC became saturated whereas DW and Nup increased exponentially in this phase. Therefore, CC was found less suitable for the estimation of traits as soon as canopy development shifted into the vertical direction ,47.On the other hand, CC clearly outperformed all other sensor methods on D1 and could still compete with most vegetation indices on D2, thus substantiating the initial hypothesis that spectral sensing is limited through soil background scattering ,27,64. TSurprisingly, the Greenseeker-NDVI exceeded all hyperspectral methods on D1. Even if all sensors were carefully positioned in the center of the sampling frame, differences in the oval field of view of the Greenseeker compared to the circular field of view of the Handyspec may have favored the Greenseeker. Compared to DW and Nup, the Greenseeker exceeded the hyperspectral indices even more for CC. Good CC prediction by the Greenseeker was also confirmed by .Among the established sensor methods, the NDVI constituted on average the best established hyperspectral vegetation index for CC estimation, but was increasingly exceeded by the NDRE for DW and Nup with ongoing plant development. Contour map analysis indicated possible improvements by 11% and 10% compared to NDRE for DW and Nup, respectively, from band combinations with a similar NIR band like the NDRE at 792 or 794 nm but a rightwards shifted red edge band (736 nm) compared to 720 nm. Thus, these indices confirm the simple ratio index R780/R740 that was previously tested for the estimation of Nup in wheat from Zadok’s stage 29 on ,66 as weWhile the REIP and R780/R740 mostly performed comparably as previously shown for later stages , the preWater-related indices combining bands in the NIR range were reported to correlate with biomass and were used for estimating the final yield ,72. In tCanopy cover is important to be estimated for evaluating early vigor. While the correlations of hyperspectral indices were lower for this trait than for DW and Nup, either spectral unmixing or Greenseeker gave the best correlations per day among the established methods. With the exception of D2, the Greenseeker-NDVI even outperformed the hyperspectral NDVI for this trait. Due to the rather weak light source of this active sensor, it may have become slightly saturated for DW and Nup for canopies with 3D structure at D4 as also found for maize . HoweverInterestingly, the early plant vigor index (EPVI) developed for detecting CC  performeSpectral unmixing was included as method that includes information of the whole spectrum which was related to extreme endmembers soil and dense vegetation. Thus, it could hold the advantage of directly returning values of fractional vegetation cover in the same range of CC. It gave convincing results with the exception of D1. The information included in the very flat canopy spectra was very similar to the soil spectrum on that day. With the soil fraction dominating the spectra at a canopy coverage of only 4.5%, distinct soil scattering was observed. Moreover, the soil surface was still rougher and more heterogeneous between plots, which added additional noise and hindered representative manual selection of reference spectra. Even when advanced endmember selection is performed, soil scattering remains a major draw-back in the VIS and NIR range . SupposiThe results substantiate the hypothesis that RGB imaging can be considered as an alternative to spectral sensing in very early growth stages to determine crop traits. While spectral measurements deliver better results for the later stages, especially at the beginning of vegetation growth they are negatively influenced by high soil reflectance. Main advantages of the RGB methodology are the reasonably low investment and maintenance costs compared to spectral crop sensors that can easily cost some US-$1000 . Further


HNF1B and cancer susceptibility, we conducted a comprehensive research synopsis and meta-analysis to evaluate associations between HNF1B variants and prostate and endometrial cancers.To provide a synopsis of the current understanding of the association between variants of OR = 1.247, p = 2.21 × 10− 77) and endometrial cancer ; the AA, AG genotypes also showed strong significant associations with the risk of prostate cancer . There was a strong significant association between rs7501939 G and the risk of prostate cancer . Strong significant association was found between rs11649743 G , rs3760511 C  and the prostate cancer risk;the GG, AG genotypes of rs11649743 also showed strong significant associations with the risk of prostate cancer . All the cumulative epidemiological evidence of associations was graded as strong.Eighteen studies totaling 34,937 patients and 55,969 controls were eligible for this meta-analysis. Four variants showed a significant association with the risk of individual cancer. Strong significant associations were found between rs4430796 A and the risk of both prostate cancer (HNF1B are associated with risk of prostate and endometrial cancer.Our study summarizes the evidence and helps to reveal that common variants of  Human cancers result in Considerable morbidity and mortality. Family history, ethnicity, lifestyle and region are potential risk factors for cancer development –4. HowevHNF1B, formerly known as TCF2) locus (on chromosome 17q12) and cancer risk [HNF1B deletion exhibit renal disease, elevated liver enzymes, and diabetes [HNF1B confers a poor overall survival in patients with multiple myeloma [Owing to advances in sequencing technologies and genome-wide association studies (GWAS), a large number of genetic variants correlated with various cancers have been identified , 9. Multcer risk –13. HNF1cer risk  and duricer risk . Patientdiabetes . HNF1B idiabetes , 17,ovardiabetes –20,endomdiabetes , 22 and  myeloma .HNF1B and the direction of their effects differ between cancer types. SNPs rs4430796 and rs7501939, are both associated with the prostate cancer risk across many ethnic groups [However, fine-mapping studies have revealed a complex genetic architecture of the HNF1B locus, demonstrating that variants of c groups . The samc groups . Yet, thc groups .HNF1B variants and cancer phenotypes, and performed a comprehensive meta-analysis, involving a total of 34,937 patients and 55,969 controls, to derive more precise estimates of the associations between HNF1B variants and susceptibility to prostate and endometrial cancers.Here, we collected data related to the associations between HNF1B variants and cancers published before July 2017. The search strategy was based on a combination of the terms (Hepatocyte nuclear factor-1 beta or HNF1B) and (cancers or tumors). The references of all computer-identified publications were searched for additional studies, and the PubMed option “Related Articles” was also used to search for potentially relevant papers. Searches were performed by two independent reviewers (Yu Tong and Yibin Wang). The language of the publications did not influence article selections.The US National Library of Medicine’s PubMed, Embase, OMIM, ISI Web of Science, and Chinese National Knowledge Infrastructure (CNKI) databases were searched in a systematic manner to retrieve all genetic association studies of HNF1B variants, and (3) the numbers of subjects possessing each allele and genotype in the cancer and control groups were available. No restrictions were set for the source of controls . Studies were excluded when: (i) they lacked sufficient information; (ii) they were published as letters to editors or conference abstracts; (iii) they were studies about cancer mortality.Studies were included if they met the following criteria. (1) the study reported original data from case-control or cohort studies, (2) the study reported alleles and genotypes for Data were extracted independently by two investigators (Yu Tong and Yibin Wang), who used recommended guidelines for reporting on meta-analyses of observational studies. The following data were extracted from the eligible studies: authors, journal title, year of publication, country of origin, selection and characteristics of cases and controls, demographic data, ethnicity of the study population, numbers of eligible and genotyped cases and controls, and genotype distributions in cases, controls, and available subgroups. Furthermore, we examined whether genotype frequencies in control groups conformed to the Hardy-Weinberg equilibrium (HWE) was determined. Any disagreement was adjudicated by a third author (Yi Qu).Minelli et al. We performed Cochran’s Q test and calculated І2 statistic to evaluate heterogeneity between studies. Harbord’s test was performed to evaluate publication bias. Potential small-study bias was evaluated by Egger’s test [metan, metabias, metacum, and metareg commands.The odds ratio was used as the metric of choice for each study. To detect overall genetic associations, allele frequencies were computed for studies reporting allele and genotype data. Pooled odds ratios were computed by the fixed effects model and the random effects model based on heterogeneity estimates. Once an overall gene effect was confirmed, the genetic effects and mode of inheritance were estimated using the genetic model-free approach suggested by r’s test . SensitiI2 < 25%, B for I2 between 25 and 50%, and C for I2 > 50%), and protection from bias . Cumulative epidemiological evidence for significant associations was thought to be strong if all three grades were A, moderate if all three grades were A or B, and weak if any grade was C.Venice criteria  were appTo determine whether a significant association could be excluded as a false positive finding, FPRP  was calculated using the method described by Wacholder et al. . FPRP < Our initial database search identified 113 potentially relevant studies. Based on a review of titles and abstracts, 55 articles were retained. The full text of these 55 articles was reviewed in detail, and 18 studies containing 36 datasets were eligible for inclusion in the meta-analysis. The specific process for identifying eligible studies and inclusion and exclusion criteria are summarized in Fig. Characteristics of the included articles are presented in Allelic associations: HNF1B rs4430796 and prostate cancer  and Caucasians  demonstrated a pattern similar to that of the full population. However, this effect was weak in the Africans . No publication bias was found in the eligible studies (Harbord’s test p = 0.253).All 15 publications were included in the evaluation of the association between the HNF1B rs7501939 and prostate cancer . No publication bias was found in the eligible studies (Harbord’s test p = 0.864).Six publications were included in the evaluation of the association between the HNF1B rs11649743 and prostate cancer . Strong significant association was observed with the prostate cancer risk .Two publications included data regarding the association between the HNF1B rs3760511 and prostate cancer. There was a strong significant association between rs3760511 and the risk of prostate cancer, and moderate heterogeneity was found among the eligible studies . No publication bias was found in the eligible studies (Harbord’s test p = 0.778).Three publications were included in the evaluation of the association between the HNF1B rs4430796 A > G and endometrial cancer . Lack of significant association was found in Africans , the Asians , and Latino and Hawaiian . No publication bias was found in the eligible studies (Harbord’s test p = 0.950).Two publications were included in the evaluation of the association between the HNF1B rs7501939 and endometrial cancer . The same pattern was observed in Caucasians , Africans , Asians and Latino and Hawaiian  (Data not shown).Two publications were included in the analysis of the association between the HNF1B rs4430796 among case and control groups is presented in Table OR1) and AG versus GG (OR2) were calculated for each study. A multivariate meta-analysis was conducted to estimate the pooled risk. There was a significantly increased risk of prostate cancer among individuals with the homozygous AA genotype  and heterozygous AG genotype .The pooled estimates were similar to those obtained after removal of the study with HW disequilibrium [OR1 = 1.524  and random effect OR2 = 1.198 .Of the 15 publications, only seven reported genotype information. The genotype distribution of the HNF1B rs11649743 among case and control groups is presented in Table OR1) and GA versus AA (OR2) were calculated for each study. Multivariate meta-analysis was conducted to estimate the pooled risk. There was a significantly increased risk of prostate cancer among individuals with the homozygous GG genotype  and heterozygous AG genotype . No between-study heterogeneity was found for the homozygous GG genotype  or for the heterozygous GA genotype .Only one publication reported genotype information for rs11649743. However, this publication included relevant data for different populations and regions. The genotype distribution for the Venice criteria were applied to evaluate these significant associations. Details of protection from bias for genetic variants significantly associated with prostate and endometrial cancer risk in meta-analyses are shown in Table P value was calculated. All associations with cancer risk had a FPRP value < 0.001. Thus, all the cumulative epidemiological evidence of associations was graded as strong.To evaluate the probability of true association with cancer risk for the nominally significant variants, FPRHNF1B variants and susceptibility to prostate and endometrial cancers. Our primary analysis revealed that, rs4430796 A, showed strong significant associations with risk of both prostate cancer  and endometrial cancer ; the AA, AG genotypes also showed strong significant associations with risk of prostate cancer . Sensitivity analyses in Caucasians demonstrated patterns similar to that of the full population. However, lack of significant association was found in Africans, which is likely due to the considerably smaller sample size. There was a strong significant association between rs7501939 A and the risk of prostate cancer ; however, lack of significant association with endometrial cancer risk was observed , and the GG, AG genotypes also showed strong significant associations with the risk of prostate cancer . Strong significant association was also found between rs3760511 C and the risk of prostate cancer . Using the Venice criteria and false-positive report probability tests, we graded all the cumulative evidence of significant associations with prostate and endometrial cancers risk as strong.To our knowledge, this is the first general overview of the association between Our findings were based on several gene-association studies, including several thousand participants, and were robust in terms of study design and sensitivity analyses. We found no evidence of publication bias or small study bias based on funnel plots. Between-study heterogeneity was found in allelic association studies (G versus A) of rs7501939, and in allelic (G versus A) of rs11649743 for prostate cancer. When HWE was examined, one study showed deviation. Our results were robust to the removal of this study.HNF1B encodes three isoforms: isoforms ; isoform A and B act as transcriptional activators and isoform C acts as a transcriptional repressor [HNF1B might modulate the risk of cancer [HNF1B were associated with the risks of both endometrial cancer in women of European background [HNF1B rs757210 is specific to serous epithelial ovarian cancer by pooling data from GWAS and follow-up genotyping; the analysis included 43 studies from the Ovarian Cancer Association Consortium [HNF1B rs7501939 was a susceptibility locus for testicular germ cell tumor [HNF1B variants predispose individuals to clear cell ovarian, endometrial, lung and prostate cancers, et al.epressor . HNF1B if cancer . Howeverckground  and prosckground  . Severackground , 46, 47.ckground  . In 201nsortium . At the ll tumor . Taken tThere are several limitations of the study. First, it is likely that some publications were overlooked although we conducted an exhaustive literature search, some relevant published studies with null results were not identified. Second, due to insufficient data, we were unable to evaluate publication bias for associations between several variants in 8q24 region and prostate and endometrial cancer. Third, a unified analysis standard across studies could not be defined for lack of raw data from the original publications. Therefore, future studies with larger sample size are warranted to confirm these associations.HNF1B variants to cancer biology, we attempted to estimate the strength of the genetic associations between these variants and prostate and endometrial cancers. This Human Genome Epidemiology (HuGE) systematic review presents strong evidence for an association between HNF1B variants and prostate and endometrial cancers, both overall and in Caucasians, Asians, Africans, and Indians, suggesting a multiplicative genetic model for variants of HNF1B among different ethnic populations. Our study results also suggest that HNF1B plays an important role in prostate and endometrial cancers, and these variations may serve as efficient and economical biomarkers for the diagnosis of prostate and endometrial cancers.Given the relevance of


Drain insertion following chronic subdural hematoma (CSDH) evacuation improves patient outcomes.To examine whether this is influenced by variation in drain location, positioning or duration of placement.We performed a subgroup analysis of a previously reported multicenter, prospective cohort study of CSDH patients performed between May 2013 and January 2014. Data were analyzed relating drain location , position , and duration of insertion, to outcomes in patients aged >16 yr undergoing burr-hole drainage of primary CSDH. Primary outcomes comprised modified Rankin scale (mRS) at discharge and symptomatic recurrence requiring redrainage within 60 d.P = .95). We found no significant differences in discharge mRS between these groups. Recurrence rates were comparable between patients with postoperative drainage for 1 or 2 d, 6.4% and 8.4%, respectively (P = .44). There was no significant difference in mRS scores between these 2 groups (P = .56).A total of 577 patients were analyzed. The recurrence rate of 6.7% (12/160) in the frontal subdural drain group was comparable to 8.8% (30/343) in the parietal subdural drain group. Only 44/577 (7.6%) patients underwent subgaleal drain insertion. Recurrence rates were comparable between subdural  and subgaleal  groups  and duration did not appear to influence recurrence rate or clinical outcomes. Similarly, drain location did not influence recurrence rate nor outcomes where both parietal and frontal burr holes were made. Further prospective cohort studies or randomized controlled trials could provide further clarification. BHCburr-hole craniostomyCSDHchronic subdural hematomamRSmodified Rankin scaleTDCtwist-drill craniostomy1 and 2 meta-analyses.3 A multicenter audit of CSDH treatment in the UK and Ireland confirmed the benefit of drain insertion in terms of symptomatic CSDH recurrence requiring reoperation within 60 d of the index surgery.4 However, that same study demonstrated significant variation in the details of surgical management of symptomatic CSDH between surgeons.The benefit of insertion of a closed subdural drainage system at the time of burr-hole drainage of symptomatic chronic subdural hematoma (CSDH) has been demonstrated in a randomized controlled trialThere is a paucity of literature examining the precise details of drain insertion and whether location, position, and duration of postoperative drainage affect outcomes. The aim of this study was therefore to determine the effect of these drain variables on clinical outcomes by performing a subgroup analysis of patients in the national audit.5 Briefly, a multicenter, prospective cohort study was conducted to determine differences in operative and perioperative strategies for managing patients with CSDH, as well as clinical outcomes. “Study participants were enrolled at 26 of the 33 UK and Ireland Neurosurgical Units (NSUs) between May 2013 and January 2014. Eligibility criteria were age >16 yr, presentation with a primary or recurrent CSDH confirmed on cranial imaging, and referral to a participating NSU”.4 Patient demographics, baseline characteristics including medical conditions and relevant medications, alongside details of pre-, intra-, and postoperative management were recorded by the local clinical teams. Symptomatic recurrence was defined as repeat surgical drainage within 60 d of index admission. “The decision to proceed with revision surgery was at the discretion of the patient's consultant neurosurgeon, based on clinical symptoms, correlated with imaging”.4 The modified Rankin scale (mRS) score and destination at discharge from the NSU, morbidity and mortality, and duration of stay in the NSU were also recorded. The following mRS was used: 0–no symptoms; 1–no significant disability ; 2–slight disability ; 3–moderate disability ; 4–moderately severe disability ; 5–severe disability ; 6–dead.The study methodology has been described in detail previously.The study protocol was approved nationally by the Academic Committee of the Society of British Neurological Surgeons. Individual participating NSUs were responsible for controlling their own contributory data, and also had local governance approvals in place. Individual patient consent was not required and therefore not sought for this study.4 Recurrence occurring on one side in a patient who underwent drainage of bilateral CSDH was considered as a single recurrence.Patients from the cohort study who were transferred to an NSU and underwent primary burr-hole craniostomy (BHC) with drain placement for either unilateral or bilateral CSDH were included in this subgroup analysis. Patients who had undergone previous drainage of an ipsilateral CSDH were excluded. Patients who underwent simultaneous drainage of bilateral CSDH were included in our analyses, as previous analyses of this dataset demonstrated no association between unilateral/bilateral CSDH and the same outcomes of interest.For those patients undergoing drain placement at the time of surgery, researchers recorded whether this had been placed in the subdural or subgaleal space (drain location). Drain position , and duration of postoperative drainage in days were recorded. Of the entire cohort, 33 patients had a single burr-hole operation with drain insertion; these patients were excluded from the drain-position analysis. However, single-burr-hole operations were included in the drain location and duration analyses, when relevant complete data were available.Chi-squared, Fisher's exact, parametric and non-parametric tests were used, when appropriate, to compare baseline clinical characteristics. These comparisons were made for each subgroup analysis . We used the Mantel–Haenszel method to examine potential confounders to the association of interest. Multiple logistic regression models were used to calculate adjusted odds ratios. Variables entered into the final multivariate analyses were those that were shown to influence the association of interest in univariate analyses, or putative factors for the outcome of interest. Putative factors entered into the multivariate analyses for recurrence were age, gender, preoperative GCS, preoperative antiplatelet medication use, and preoperative warfarin use. In addition to these factors, dichotomized admission mRS and bed rest were entered into multivariate analyses for functional outcome. Functional outcome was the discharge mRS categorized into favorable (mRS 0-3) and unfavorable (mRS 4-6) outcomes. Likelihood ratio tests were used to determine the model of choice. Patients with missing data were excluded if the missing data were relevant to that particular analysis. Stata version 13.0  was used for all analyses.Data from 1205 patients were collected in the national audit. Of these, 577 patients had burr-hole drainage of their CSDH with drain insertion and were identified for inclusion in our subgroup analysis; details of their baseline, perioperative, and operative/postoperative characteristics are detailed in Table P = .95, Table P = .48, Table The majority of patients in our analysis underwent subdural drain insertion . The recurrence rate of 7.7% (41/533) in the subdural drain group was not significantly different from 9.1% (4/44) in the subgaleal group (P = .04). Complications according to the duration of drain are shown in Table Multivariate analyses revealed that when adjusting for the pre-specified variables that affect recurrence rate and functional outcome, there appeared to be no correlation between different drain location/position and outcomes Tables  and 4. AP = .44, Table P = .56, Table P = .09, Table Only 4.9% of patients had drain insertion for at least 3 d, so were excluded from the subsequent analysis Table . Analysi6 The median time to recurrence in the present study agrees with that reported previously.7The location, position, and duration of drain insertion following burr-hole drainage of a chronic subdural hematoma do not affect symptomatic recurrence requiring repeat surgery, or functional outcome. This subgroup analysis comprises the largest study performed on drain position and duration in CSDH. The symptomatic recurrence rate we observed at 60 d may be underestimated if there were even later recurrences; however, previously published data suggest that recurrence is most likely within this time period.4Where variation in practice does not impact on patient outcomes, there may be opportunities to simplify care pathways, reducing costs, and enhancing patient experiences. There are many operative and perioperative variables in the management of a patient with CSDH. In our previous multicenter cohort study, we demonstrated that drain insertion was an important predictor of outcomes. Other common variations in practice such as duration of postoperative bed rest and prescription of high-flow oxygen were not.1 specified drain placement in the subdural space through either burr hole, kept in a dependent position, and removed at 48 h. They demonstrated significantly lower recurrence rates and improved 6-mo outcomes when compared to no drain use.1 Follow-up data have also demonstrated better long-term survival in the drainage group.8 However, the authors did not indicate the length of drain to be inserted intracranially, and postoperative imaging was not collected to confirm the position of the drain. Similarly, we did not confirm the drain position radiologically in our cohort and this analysis was dependent on details recorded in the operative notes.In their randomized controlled trial of drain use, Santarius and colleagues9 and Zumofen et al10 reported retrospective medium-sized case series of extracalvarial  drain insertion following BHC. Neither study had a subdural drain group for comparison, but the authors both reported recurrence rates comparable with the published literature. In a prospective, non-randomized study, Chih et al11 demonstrated no significant difference in recurrence rate and functional outcome. A small prospective randomized study purported no difference in recurrence rates but a better functional outcome in the subperiosteal drain group; this may have been confounded by the superior preoperative average mRS score in the subperiosteal group.12 By contrast, a retrospective single-center comparative study reported a higher re-operation rate for symptomatic recurrence in the subgaleal drain group compared to the subdural drain group, but there was a non- significant tendency to less-serious complications and lower 1 yr mortality in the subgaleal drain group.13 Similarly, Chih et al11 observed a non-significant increase in complications in the subdural drain group.Gazzeri et al14Perhaps the precise location of drain insertion in relation to the burr hole is not important. Without a standard definition of ‘subdural’ and ‘subgaleal’ drain position, it is possible that the drain positions may not differ that much; some drains described as being positioned in the subdural space may, in fact, be inserted a little distance beyond the burr hole, similar to drains positioned over the burr hole. Proponents of subgaleal drains argue that by avoiding instrumenting the subdural space, there should be a reduced risk of intracranial hemorrhage, brain injury, empyema, and epilepsy. We observed no significant difference in seizures between subdural and subgaleal drain groups. However, our results should be interpreted with caution given the small numbers of subgaleal drains used during the study period, and likely selection bias, as well as lack of prespecified data collection on intracranial hemorrhage and empyema. An adequately powered randomized controlled trial of subdural vs subgaleal drains is needed, one of which is underway in Switzerland.16 Our results suggest that it does not matter through which burr hole a drain is placed in terms of recurrence and functional outcome, although we observed a higher risk of new neurological deficit when the drain was placed via the frontal burr hole. It is difficult to ascertain the clinical significance of this finding because of the small number of events reported and the reporting of neurological deficit was not part of our primary outcome, therefore defined broadly and may be subjected to reporting bias. We are also unable to comment upon drain direction and ultimate tip position. Several small retrospective studies demonstrated no impact of the position of the intracranial subdural drain on recurrence rates.17-19Previous studies have also examined whether there is an advantage to frontal or parietal drain placement. Two small retrospective studies of patients undergoing BHC reported lower recurrence rate when the drain tip is placed in the frontal position within the subdural space.20 retrospectively compared recurrence rates after BHC in those drained for 2 to 4 d vs 5 to 7 d. They reported a significantly lower recurrence rate in the 5 to 7 d group (3.3% vs 15.6%), although the difference in actual reoperation rate between groups was much less stark (5% vs 2%). Yu et al21 reported significantly different recurrence rates in their retrospective series of 97 patients undergoing BHC (16.3% for patients drained <3 d vs 1.3% in those drained ≥3d), although presenting GCS scores were not recorded and may have differed between 2 groups. A randomized controlled trial comparing 48 h vs 96 h of postoperative drainage after twist-drill craniostomy (TDC) demonstrated no difference in recurrence rates, but increased mortality and complications and in the 96 h group.22 Sindou et al23 also performed a small randomized study comparing the same drainage durations in TDC and reported similar results. Jeong Si et al16 found no difference in recurrence rate when comparing <2 d and ≥2 d in BHC. Given the size and design of these studies, it is difficult to ignore the findings of the high-quality drain study from Santarius and colleagues1 that utilized a 48 h drainage period.8 The question of drain duration merits further study.We observed no apparent advantage to drainage for more than 24 h and prompt drain removal may facilitate early mobilization, which, in turn, could reduce morbidity and improve functional outcomes. Previous studies examining the impact of duration of postoperative drainage on CSDH recurrence and functional outcomes are contradictory. Kale et alRecurrence rate and functional outcome after drainage of primary CSDH by BHC does not appear to be affected by drain location, position, or duration of drainage. Choice of drain position and duration will be guided by surgeon preference and intraoperative findings, but variation in practice may not influence outcomes. Well-designed and adequately powered randomized controlled trials will help interrogate questions of drain position, location, and duration further.Dr Hutchinson is supported by a Research Professorship from the National Institute for Health Research (NIHR), the NIHR Cambridge Biomedical Research Centre, a European Union Seventh Framework Program grant , and the Royal College of Surgeons of England. Dr Kolias is supported by a Clinical Lectureship, School of Clinical Medicine, and University of Cambridge. The authors have no personal, financial, or institutional interest in any of the drugs, materials, or devices described in this article.


MLH1 mutation for a patient diagnosed with a rectosigmoid adenocarcinoma at age 31.Lynch syndrome (LS) is caused by germline mismatch repair (MMR) gene mutations. De novo MMR gene mutations are rare, and somatic mosaicism in LS is thought to be infrequent. We describe the first case of somatic mosaicism by a de novoTwelve years after initial colorectal cancer diagnosis, tumor tissue of the patient was tested with sensitive next generation sequencing (NGS) analysis for the presence of somatic MMR mutations.MLH1 mutation ) was detected, which was also present at low level in the blood of the patient. In both parents, as well as the patient's sisters, the mutation was not present.In tumor tissue, an inactivating We show that low‐level mosaicism can be detected by using high‐coverage targeted NGS panels on constitutional and/or tumor DNA. This report illustrates that by using sensitive sequencing techniques, more cases of genetic diseases driven by mosaic mutations may be identified, with important clinical consequences for patients and family members. Furthermore, Sourrouille et al. (MSH2 (OMIM 609309), which was detected in normal colonic tissue but not in the blood. This mosaic MSH2 mutation apparently had germline transmission since this mutation was also present in the patient's son. Here, we report the first case of somatic mosaicism by a de novo MLH1 mutation.Heterozygous germline mutations in MMR genes cause Lynch syndrome (LS), an autosomal dominant condition which predisposes to various types of cancer including colorectal cancer (CRC) and endometrial cancer  of DNA isolated from blood showed no indication for the presence of a germline mutation. The bladder tumor of the index patient's uncle was also examined but did not show any signs of MSI (IHC was not possible). Based on these results, the patient was considered to be Lynch‐like. Therefore he and his sisters underwent colonoscopy every 2 years, no polyps were identified. Additionally, surveillance for extra‐colonic tumors was advised to him and his sisters. One sister decided to have her ovaries and uterus preventively removed at 46 years of age. In both organs, no signs of neoplastic growth were observed.Because of his young age, he was referred for genetic counseling to the Familial Cancer Clinic. Immunohistochemistry (IHC) of the MMR proteins was performed on the tumor tissue, which showed normal nuclear expression of MSH2 and MSH6 but absence of nuclear expression of MLH1 and PMS2. Molecular analyses showed MSI without Recently, all Lynch‐like patients of the Familial Cancer Clinic at the Netherlands Cancer Institute are being evaluated for somatic MMR gene mutations, as the presence of a somatic MMR gene mutation can indicate a sporadic origin of the tumor. Therefore, last year the index patient visited the Familial Cancer Clinic again, 12 years after the initial CRC diagnosis. Subsequently, the tumor was tested with next generation sequencing (NGS) for the presence of somatic MMR mutations.22.1The index patient was counseled by the Familial Cancer Clinic and provided informed consent.2.2MLH1 (NM_000249.3), MSH2 (NM_000251.2), MSH6 , and PMS2 , with a coding sequence coverage of 97%, 94%, 95%, and 81% respectively, and hotspots for BRAF , POLD1 , and POLE . Furthermore, single nucleotide polymorphisms in and around the MLH1, MSH2, MSH6, and PMS2 genes are included to detect copy number variations, as previously described  tissue, after which proteinase K and 5% Chelex 100 resin was added for DNA isolation, as previously described .2.3MLH1 exon 6 was performed using standard procedures.DNA isolation from blood of the index patient and his two sisters was performed using DNAzol (Thermo Fischer Scientific). DNA from normal FFPE tissue of the deceased parents was isolated using QIA Amp DNA purification kit (Qiagen). Sanger sequencing for 3MLH1 mutation ; see Figure MLH1 allele was seen, indicating biallelic MLH1 inactivation. This explains the MSI phenotype and the absence of nuclear expression of MLH1 and PMS2 in the tumor cells. Additionally, five missense variants of unknown significance were detected in tumor tissue only, one in MSH2 ) and four in MSH6 , c.1730G>A; p.(Arg577His), c.2419G>A; p.(Glu807Lys) and c.3163G>A; p.(Ala1055Thr)).In the tumor, an inactivating MLH1 mutation was also detected by NGS at low frequencies in DNA isolated from several normal tissues, including smooth muscle near the tumor, epithelium from the small intestine and lymph nodes without metastasis , enabling detection of low‐frequency mutations.When routine mutation analysis of constitutional DNA from patients with a clear clinical suspicion for a tumor syndrome is negative, one should consider to perform high‐coverage targeted NGS of the indicated genes to detect possible mosaicism. Furthermore, sequencing of tumor tissue can guide detection of a low‐level mosaic variant in the germline. This diagnostic strategy will result in improved health care both for the index patient and his or her family members.The authors declare no conflict of interests.


A single mild traumatic brain injury (mTBI) typically causes only transient symptoms, but repeated mTBI (RmTBI) is associated with cumulative and chronic neurological abnormalities. Clinical management of mTBI is challenging due to the heterogeneous, subjective and transient nature of symptoms, and thus would be aided by objective biomarkers. Promising biomarkers including advanced magnetic resonance imaging (MRI) and plasma levels of select proteins were examined here in a rat model of RmTBI. Rats received either two mild fluid percussion or sham injuries administered five days apart. Rats underwent MRI and behavioral testing 1, 3, 5, 7, and 30 days after the second injury and blood samples were collected on days 1, 7, and 30. Structural and diffusion-weighted MRI revealed that RmTBI rats had abnormalities in the cortex and corpus callosum. Proteomic analysis of plasma found that RmTBI rats had abnormalities in markers indicating axonal and vascular injury, metabolic and mitochondrial dysfunction, and glial reactivity. These changes occurred in the presence of ongoing cognitive and sensorimotor deficits in the RmTBI rats. Our findings demonstrate that RmTBI can result in chronic neurological abnormalities, provide insight into potential contributing pathophysiological mechanisms, and supports the use of MRI and plasma protein measures as RmTBI biomarkers. A single mTBI can result in a range of neurological impairments that are typically transient2. On the other hand, repeated mTBIs (RmTBI), which are particularly common in contact sports and military settings, have been associated with significant and lasting neurological abnormalities, and have been indicated as a risk factor for developing various neurodegenerative diseases5.Mild traumatic brain injuries (mTBI), such as concussions, are induced by biomechanical forces acting on the brain, and are a common and significant health problem worldwide7. As such, the identification of objective biomarkers for these injuries has become a research priority in recent years10. Previous preclinical and clinical findings have found that advanced magnetic resonance imaging (MRI) and blood-based protein measures are promising and clinically applicable mTBI biomarkers13. For instance, diffusion-weighted MRI (DWI) methods, such as diffusion tensor imaging (DTI) and tractography, detect the diffusion of water molecules within the brain and are sensitive to pathophysiological changes, such as axonal injury, that can be induced by mTBI16. Blood-based protein biomarkers are also capable of detecting a number of pathobiological changes that may occur following mTBI, including metabolic, neuronal, axonal, glial, inflammatory, and vascular changes20. Of particular relevance to this study, we have previously found that a single mild fluid percussion injury (mFPI) in rats results in abnormalities detectable by both DWI and plasma protein measures, and that these changes persisted longer than the cognitive abnormalities that recovered by day five post-mFPI12.The heterogeneous and subjective nature of mTBI and RmTBI symptomatologies present serious challenges in the clinical management of these injuries22. In particular, investigating the effects and evolution of clinical RmTBI present additional challenges and complexities  than studies of a single mTBI. Furthermore, as RmTBI is quite common in athletes and soldiers, it is important to understand how RmTBI might influence biomarkers differently than an initial mTBI. Therefore, in this study we used the repeated mFPI (RmFPI) rat model to determine changes detected by MRI, select plasma protein biomarkers, and neurobehavioral measures at different recovery times after RmTBI.Although there is growing evidence that MRI and blood-based protein measures can detect changes after a single mTBI, how RmTBI affects these measures and how they relate to the functional consequences of RmTBI remains largely unknown relative to the growing understanding of a single mTBI. This is in part due to the difficulties in rigorously studying the effects of RmTBI and characterizing biomarkers purely in the clinical settingForty-eight male Long-Evans rats were purchased from Monash animal research services . All rats were 8–12 weeks of age and weighed 250–300 g at time of injury. Following surgery, rats were housed individually under a 12 h:12 h light/dark cycle with ad libitum access to water and food for the duration of the study. All experimental procedures were approved by The University of Melbourne and The Florey Institute of Neuroscience and Mental Health animal ethics committees, and complied with the guidelines of the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. It is important to note that data collection for this study was done in conjunction with another study that examined behavior, blood, and MRI biomarkers after a single mFPI [12]. This was done in an effort to reduce animal usage, and therefore only a single group of sham-injured rats was used. Consequently, there is some overlap between the sham data presented in this paper and our previous paper [12].25, while RmFPI that are separated by 5 days induce cumulative and chronic pathological and behavioral changes29. These abnormalities resemble the neurological consequences that mTBI and RmTBI patients may experience, and provides the rationale for the use of the RmFPI model in this study.Our laboratory, and others, have previously demonstrated that a single mFPI induces temporary behavioral and pathophysiological abnormalities29. Briefly, under isoflurane induced anesthesia a craniotomy (5 mm diameter) was performed  to expose the intact dura and a hollow injury cap was attached to the skull over the craniotomy. Rats were then attached to the fluid percussion device via the head cap. At the first sign of hind-limb withdrawal to a toe-pinch, rats received a fluid percussion pulse of 1–1.5 atm before being disconnected from the device. Sham-injury rats underwent the same procedures except the fluid injury pulse was not administered. Topical antibiotic ointment was then applied, and a sterile plug was inserted into the injury cap to seal the craniotomy. Five days following the initial injury, rats were anaesthetized for their second mFPI or sham injury. The plug was removed from the cap, the cap was filled with saline and the rat was connected to the FPI-device. At first response of hind limb withdrawal sham rats were removed from the device and RmTBI rats received an FPI 1–1.5 atm.All of the procedures used were based on standard protocols as previously described32.Acute injury measures were monitored after each injury, as shown in Table Following their last assigned mFPI or sham injury, rats were allocated to separate behavior  or MRI  cohorts. The behavior cohort underwent behavioral testing on days 1 (D1), 3 (D3), 5 (D5), and 7 (D7) after the second injury before being euthanized on D7 for blood collection. The MRI cohort underwent MRI at D1, D3, D5, D7 and D30 after the second injury before being euthanized on D30 for blood collection. Note that the MRI cohort also underwent behavioral testing on the day prior to their D30 MRI. This study design allowed us to maximize the amount of data collected on corresponding post-injury days, and also avoided the confounding factor of anesthesia (required for MRI) on behavioral outcomes. An additional 14 rats  were euthanized at D1 for blood collection/plasma protein analysis. Thus, plasma samples were available for D1, D7, and D30 post-injury.12. The water maze apparatus consisted of a circular tank (163 cm in diameter) filled with water (29 °C), and a hidden acrylic escape platform (10 cm diameter) submerged 2 cm below the water surface in one of the quadrants of the pool. Unique visual cues were positioned at the north, south, east and west quadrants. Both the position of the escape platform, and the visual cues, were randomly assigned each day to maintain novelty throughout the course of the experiment. Rats completed four trials per testing day, with the entry position randomized between north, south, east and west for each trial. If the rat failed to find the platform in the allotted minute, then they were placed on the platform for 15 s. Ethovision behavioral tracking software analyzed video from an overhead camera positioned at the center of the tank. Search time , as well as swim speed , were quantified for each trial.A researcher blinded to experimental conditions conducted all behavioral testing. Cognitive function was assessed using the water maze12. Baseline training was performed prior to surgery and consisted of five successful trials on a 4 cm wide beam, followed by five successful trials on a 2 cm wide beam. On each day of testing, rats completed ten trials on the 2 cm wide beam, and the time taken to traverse the beam and the numbers of slips and falls were recorded. The maximum time allowed per trial was 60 s, which was also assigned to rats that fell.Sensorimotor function was assessed using a 1 m long, 2 cm wide elevated wooden beam as previously described12. Ethovision behavioral tracking software  analyzed video from an overhead camera positioned at the center of the field. A single 5 minute trial was performed on each day of testing with the rat positioned in the center of the field. The total distance travelled was analyzed as a measure of locomotion.Locomotor activity was assessed using a circular open field  as previously described12. The maze consists of both open and closed arms, 110 cm long and 12 cm wide, perpendicular to each other, and dividing each in half to form a cross. Fifty cm high walls enclosed the closed arms while the open arms contained no walls. A single 5 minute trial was performed on each day of testing with the rat initially positioned at the center of the cross facing an open arm. Ethovision behavioral tracking software  analyzed video from an overhead camera positioned at the center of the cross. The percentage of time spent within the open arms , as well as the total distance travelled , were calculated.Anxiety-like behavior was assessed using an elevated plus maze, as described previously12. Anesthesia was induced using 4% Isoflurane in a 1:1 mixture of medical grade air and oxygen. Following loss of consciousness, rats were positioned on a purpose-built animal holder with ear and bite bars to immobilize the head, and a nose cone to deliver Isoflurane to maintain anesthesia. An anatomically shaped 4-chanel surface receive coil was positioned directly over the rat’s head and affixed to the animal holder which was then inserted into an 86 mm volume transmit coil for imaging with a Bruker 4.7/30 MRI.The MRI cohort underwent imaging on D1, D3, D5, D7 and D30 as described previously2*-weighted structural images were acquired using a 2D multi-gradient echo sequence with the following imaging parameters: repetition time (TR) = 4400 ms; number of echoes = 8; echo times (TE): 7.5, 15, 22.5, …, 60 ms; field of view (FOV) = 28.8 × 28.8 mm2; matrix size = 160 × 160; number of slices = 64; and slice thickness = 180 μm giving an isotropic spatial resolution of 180 × 180 × 180 μm3.T2; matrix size = 128 × 128; number of slices = 24; slice thickness = 600 μm; diffusion duration (δ) = 3.5 ms; diffusion gradient separation (Δ) = 14 ms; and b-value = 1200 s/mm2. Diffusion weighting was performed in 81 directions and 8 non-diffusion-weighted (b0) images were also acquired.DWI was performed using a 2D echo planar-based sequence with the following imaging parameters: TR/TE = 6,000/35 ms; FOV = 25.6 × 25.6 mm2*-weighted echo images were averaged across echoes and templates generated at each time point for each cohort using Advanced Normalization Tools . The resulting templates were then combined, again with ANTs, to create a study template. To assess for RmTBI-induced atrophy/hypertrophy, each rat’s mean echo image was then registered to the study template and a tensor-based morphometry (TBM) analysis was performed as previously described33.Thttp://dti-tk.sourceforge.net/) as described previously12. Regions of interest (ROI) were traced on the study template to identify the ipsilateral and contralateral corpus callosum. For each ROI, the mean FA, radial diffusivity (RD), axial diffusivity (AD) and trace (TR) were calculated using FSLstats, part of the FMRIB software library (https://fsl.fmrib.ox.ac.uk/fsl/fslwiki/FSL).Diffusion tensor images were calculated for each rat and registered to a study template using the Diffusion Tensor Imaging ToolKit 12. Briefly, fibre orientation distribution images were calculated from normalized diffusion images using constrained spherical deconvolution with a group average single-fibre response function. Whole brain tractograms were generated from 2 million streamlines and registered to the study DTI template to normalize both the length and spatial location of the streamlines. Three track-weighted images were generated from the normalized tractograms: average pathlength map (APM), reflecting the average length of all streamlines passing through each voxel34; mean curvature map, representing the average curvature of all tracks passing through each voxel12; and the track density image (TDI), which maps the total number of streamlines passing through each voxel35. As with the DTI measures, the mean of each track-weighted image was calculated for the ROIs.Tractography was performed as described previously using the MRtrix software package . The chest cavity was then opened and blood was collected into a BD Vacutainer K2 EDTA (K2E) Plus Blood collection tube via cardiac puncture. The tubes were then centrifuged at 6,000 g for 15 min at room temperature, and plasma was pipetted into 0.5 mL aliquots, flash-frozen in liquid nitrogen, and stored at −80 °C until use.36, tau protein 37, vascular endothelial growth factor 38, 4-hydroxynonenal Michael adducts 39, NF-H 40, neuron-specific enolase 41, glial fibrillary acidic protein 42, and S100 calcium binding protein β subunit 43, were assayed using reverse phase protein microarray (RPPM).Plasma levels of ceruloplasmin 12. Frozen plasma samples were thawed on ice and diluted 1:10 with Dilution Buffer . Samples were then transferred to a JANUS Varispan Integrator and Expanded Platform Workstation  to perform 1:1 serial dilutions using Dilution Buffer in 384 well microarray plates . An equal amount of 2x PBS Buffer  was added to the volume of liquid in each well. The microarray plates were subsequently transferred into an Aushon 2470 Arrayer  where plasma samples were printed onto ONCYTE AVID nitrocellulose film slides . The Aushon 2470 Arrayer was set up with 16 pins and programmed for 2 depositions per spot. The spot diameter was set to 250 nm with spacing between dots at 500 nm on the x-axis and 375 nm on the y-axis. Wash time was set to 2 s without delays. After overnight desiccation at 4 °C, the slides were blocked with a solution of 1x TBS, 5% non-fat dry milk, and 0.1% Tween-20.Sample preparation, printing, scanning, and data analysis for RPPM were performed as described previouslySlides were then incubated with the primary antibody solutions and a cover slip  overnight at 4 °C. The primary antibodies were diluted in antibody incubation buffer  and used in the following dilutions: ceruloplasmin , NF-H , tau , VEGF , 4-HNE , NSE , GFAP , and S100β . The following day, slides were washed three times with TBST and then incubated with the appropriate secondary antibody solutions for 1 h at room temperature. The secondary antibodies Alexa Fluor 790 goat anti-rabbit , 680 rabbit anti-goat , and 680 goat anti-mouse   were used at a 1:20,000 dilution. After three thorough washes with TBST followed by a single wash with 1x TBS, the slides were air dried and subsequently scanned in an Innopsys InnoScan 710 IR microarray scanner .Scanner fluorescence data were imported into a Microsoft Excel-based bioinformatics program. After correcting for local background noise, points indiscernible from background were excluded . Net intensity vs. dilution was plotted on a log-log scale. A five-parameter logistic (5PL) master curve was fitted to the slide. Using this master curve, outliers were excluded using the False Discovery Rate method (Q = 0.01) and the master curve is further refined. Each local block of samples is fit individually, using master curve values as the initial conditions for the local curve fit. The asymptotic maximum and minimum, Hill-Slope, and asymmetry constant were shared between samples. The slope of the linear portion of the logistic curve was calculated and the line extrapolated back to zero , assessing the amount of protein expressed. Data  are presented as the mean ± S.E.M.p ≤ 0.05.With the exception of the TBM analysis  and the acute injury measures presented in Table 1, 71 = 40.51, p = 0.0001) and recovery time  on the measure of platform search time , suggesting that RmTBI did not affect anxiety-like behavior.For the elevated plus maze, two-way ANOVA detected significant main effects of injury .For TDI, two-way ANOVA detected a significant injury x time interaction in the ipsilateral corpus callosum  and tractography  metrics in the corpus callosum suggestive of axonal injury16. The brain damage detected by MRI may help account for the behavioral deficits seen in the RmTBI rats. For example, the RmFPI impact site is adjacent to the sensorimotor cortex, and the observed cortical atrophy may explain the persistent sensorimotor deficits on the beam task45. Furthermore, the structural changes in the hippocampus, which plays significant roles in memory, cognition, and spatial navigation, may contribute to the observed deficits in the water maze task46.The aim of this study was to assess the utility of MRI and select plasma protein biomarkers, as well as neurobehavior measures, to detect acute, sub-acute, and long-term changes in rats that received two mTBIs. Consistent with previous studies that have administered RmFPI separated by a five-day inter-injury time40, astroglia damage (increased GFAP)47, vascular irregularities (decreased VEGF)38, oxidative stress (increased 4-HNE)39, and metabolic dysfunction (increased ceruloplasmin)36. These findings support the use of plasma protein biomarkers to identify changes following RmTBI, and to provide insight into the mechanisms that underlie the brain damage observed in these rats. Elevated levels of circulating NF-H is considered a marker of axonal injury48. The current findings that NF-H levels remain elevated until at least D30 post-RmTBI are consistent with the changes in DWI measures, and together indicate that RmFPI induces lasting and perhaps progressive axonal injury. VEGF stimulates angiogenesis and neurogenesis49. Previous studies have found that inhibition of VEGF signaling results in increased cell death after TBI50, while treatment with VEGF is neuroprotective51. Therefore, the present findings of a lasting decrease in VEGF in the presence of behavioral impairments and brain injury are consistent with the notion that VEGF may play a beneficial role in recovery after neurotrauma38. Ceruloplasmin is involved in the transport of free iron across the cell membrane and in iron metabolism53. Increased levels found in the RmTBI rats may be a response to increased iron levels that can be induced by TBI due to bleeding  and/or disruptions in ion homeostasis56. Excess iron levels can also trigger neuroinflammation and the production of reactive oxygen species58, both of which are mechanisms implicated in neurodegeneration and may help explain the observed increases in 4-HNE59.RPPM analysis of plasma protein biomarkers found changes in RmTBI rats indicative of axonal injury (increased NF-H)As alluded to in the introduction, we previously investigated behavior, MRI, and plasma protein outcomes after a single mFPI at the same recovery times studied in the present study [12]. For the behavior measures, the only statistically significant behavioral difference found in the previous study was that a single mFPI resulted in water maze deficits at day 3 post-injury, and these resolved to sham-injured levels by day 5. This is in stark contrast to the persisting water maze and beam deficits observed in the RmTBI rats in the current study. For the MRI outcomes, a single mFPI did not result in significant volumetric differences in the cortex, hippocampus, corpus callosum, or lateral ventricles compared to sham-injured, but did induce changes on DWI measures at days 1, 3, 5 and 30 post-injury. In comparison, the present study found evidence of structural MRI changes after RmTBI, with a midline shift as well as deformation of the ipsilateral cortex, hippocampus, and corpus callosum. On the DWI measures, there was evidence of differences between the RmTBI and sham groups at each of the recovery times, and there appears to be more robust differences on DWI measures at day 30 post-injury in the RmTBI rats relative to the single mFPI. However, it is important to consider that the analyses of the MRI measures differed between the studies, with the present paper using updated methodologies. For the plasma protein markers, a single mFPI was previously found to induce increased levels of ceruloplasmin and NF-H at day one post-injury, decreased tau at day seven post-injury, and decreased VEGF at days seven and thirty post-injury. Here, we also found changes on the same markers, though these changes were present across each of the recovery times. Additionally, the current study found a statistically significant increase of 4-HNE in the RmTBI rats compared to sham-injury, which was not found in the single mFPI study.12, may also provide insight into mechanisms that underlie increased cerebral vulnerability (ICV) to RmTBI. Growing evidence suggests that the long-term deleterious effects of RmTBI may be due to sustaining a RmTBI before the brain has recovered from the initial mTBI and is in a period of ICV61. We, as well as others, have previously found that a single mFPI results in transient behavioral abnormalities, with no evidence of major structural brain damage or significant neuronal loss62. Of particular relevance, we previously found that the behavioral deficits induced by a single mFPI recover to sham levels by day five post-injury , and that conventional structural MRI was unable to detect differences between the single mFPI and sham groups at that time12. However, the single mFPI rats showed changes on advanced DWI  and plasma levels of protein biomarkers  that persisted beyond the resolution of behavioral changes12. Accordingly, our current findings of lasting neurological abnormalities after a second mFPI, given in the presence of DWI and plasma biomarker changes induced by the first mTBI, may provide insight into pathophysiological changes that underlie ICV60. For example, angiogenesis post-TBI may help restore the supply of blood and oxygen to damaged tissue63. If this process has been compromised in mFPI rats, as decreased VEGF suggests, the brain may be more vulnerable if exposed to a subsequent insult.Findings from the current RmTBI study, combined with findings from our previous single mFPI study64, is beneficial after mTBI66 and it would be of interest to investigate the precise biological mechanisms that drive this effect. The DWI and NF-H biomarker findings suggest axonal injury as a central pathophysiology in RmTBI, and a number of biochemical mechanisms may contribute to this. For example, oxidative stress and neuroinflammation can contribute to axonal injury, and findings from the current  and previous studies suggest that oxidative stress and neuroinflammation are present after mTBI28. As such, interventions targeting these mechanisms may mitigate ICV and axonal injury in RmTBI. With that said, it is important to note that this discussion on underlying mechanisms/ICV and therapies is largely speculative, as a limitation of the current study is the lack of outcomes that directly assessed neuropathology. Although the biomarker methods used in this study are translatable to the clinical setting, ideally the changes in MRI and plasma protein measures would be compared against direct measures of neuropathophysiology. For example, histological studies that examined axonal integrity  and neurovascular injury would greatly complement the current biomarker findings. Taken together, future studies are still required to determine the precise neuropathological mechanisms that underlie the biomarker findings and ICV; to ascertain whether the biomarkers can be used to determine safe return to play/duty decisions and vulnerability to develop neurodegenerative changes; and to investigate intervention options to improve recovery.The current findings may also provide insight into potential therapeutic avenues. The VEGF and ceruloplasmin findings suggest neurovascular injury, therefore interventions that upregulate angiogenesis or VEGF may mitigate ICV and the consequences of RmTBI. Notably, there is recent evidence that exercise, which is known to increase VEGF and angiogenesis amongst other things69. Furthermore, the physical properties of the rodent brain relative to the human brain likely influences the nature of the brain injury. For example, the rat brain has a smooth surface as compared to the human brain with its many sulci and gyri. As such, the smooth brain of the rat may serve as a plate of compression on underlying structures whereas the gyri and sulci in humans may disperse the concussive forces in a different manner. These factors may account for the exacerbated acute and sub-acute brain injury observed in the RmTBI rats in this study relative to what would be expected in humans who have sustained RmTBI. Although the use of the mFPI model is justifiable by previous studies showing that single and repeated mFPI can result in behavioral and pathophysiological changes70, in a manner that is largely consistent with what occurs in the clinical setting2, newer models of mTBI/concussion that avoid invasive procedures and anesthetics are now being developed73. For example, our group and others have recently developed an awake closed head injury model of concussion that avoids many of the abovementioned confounding variables73. Therefore, while the current findings provide important insights into potential biomarkers for RmTBI, future studies should strive to replicate the current findings in next generation mTBI models, as well as in human participants21.A limitation of this study that should be acknowledged is that the mFPI model of mTBI is not optimal as it requires the use of anesthetics and surgery , both of which have the potential to confound behavior, MRI, and plasma protein resultsIn conclusion, how RmTBI affects MRI and plasma protein biomarkers, and how these relate to the functional consequences of RmTBI, is not well understood. To that end, this study has characterized the changes in brain microstructure detected by MRI, plasma levels of protein biomarkers using proteomics, and neurobehavioral outcomes at different recovery times after RmTBI in rats. We found that rats given RmTBI had persistent cognitive and sensorimotor deficits, abnormalities on structural MRI and DWI measures, as well as increased plasma levels of protein markers indicative of axonal injury, glial reactivity, oxidative stress, vascular irregularities, and mitochondrial and metabolic dysfunction. Although future studies are required, these findings provide additional evidence that RmTBI can result in long-term neurological deficits, offer insight into the pathophysiological mechanisms that contribute to these consequences, and support the use of MRI and plasma proteomics to objectively detect abnormalities after RmTBI.


Nicotine exposure during the fetal and neonatal periods [developmental nicotine exposure (DNE)] is associated with ineffective upper airway protective reflexes in infants. This could be explained by desensitized chemoreceptors and/or mechanoreceptors, diminished neuromuscular transmission or altered synaptic transmission among central neurons, as each of these systems depend in part on cholinergic signaling through nicotinic AChRs (nAChRs). Here, we showed that DNE blunts the response of the genioglossus (GG) muscle to nasal airway occlusion in lightly anesthetized rat pups. The GG muscle helps keep the upper airway open and is innervated by hypoglossal motoneurons (XIIMNs). Experiments using the phrenic nerve-diaphragm preparation showed that DNE does not alter transmission across the neuromuscular junction. Accordingly, we used whole cell recordings from XIIMNs in brainstem slices to examine the influence of DNE on glutamatergic synaptic transmission under baseline conditions and in response to an acute nicotine challenge. DNE did not alter excitatory transmission under baseline conditions. Analysis of cumulative probability distributions revealed that acute nicotine challenge of P1–P2 preparations resulted in an increase in the frequency of nicotine-induced glutamatergic inputs to XIIMNs in both control and DNE. By contrast, P3–P5 DNE pups showed a decrease, rather than an increase in frequency. We suggest that this, together with previous studies showing that DNE is associated with a compensatory increase in inhibitory synaptic input to XIIMNs, leads to an excitatory-inhibitory imbalance. This imbalance may contribute to the blunting of airway protective reflexes observed in nicotine exposed animals and human infants. The number one risk factor for sudden infant death syndrome (SIDS) is maternal smoking. While the use of nicotine delivery devices such as e-cigarettes is increasing among women of childbearing age, reflecting the belief that the use of nicotine alone is safer than tobacco, SIDS deaths are not decreasing, suggesting that nicotine is the link between maternal smoking and SIDS. Here, we show that perinatal nicotine exposure alters a major motor pathway responsible for upper airway patency during sleep. We also introduce an animal model that is well suited to probing the mechanisms underlying the link between maternal nicotine consumption and SIDS, and a phenotype that nicely models key aspects of the events believed to give rise to SIDS.Hypoglossal motoneurons (XIIMNs) innervate the muscles of the tongue, which are critically important in the maintenance of airway patency during breathing . The brein vivo by recording the breathing-related tongue muscle EMG before, during and after a period of airway occlusion in lightly anesthetized neonatal rats. Next, since hypoxia and hypercapnia (as occurs during airway occlusion) is associated with increased acetylcholine release  were anesthetized with a subcutaneous injection of ketamine (25 mg/kg), xylazine (8.0 mg/kg), and acepromazine (1 mg/kg) and an Alzet 1007D mini-osmotic pump (Alzet Corp.) was implanted subcutaneously on gestational day 5. A subcutaneous injection of buprenorphine (0.5 mg/kg) was given to control postoperative pain. The 28-d pump exposes the pup via the placenta throughout the remainder of gestation (∼16 d), and via breast milk after birth, and these successive pre and postnatal exposures define DNE. The pump was loaded to deliver an average dose of 6 mg/kg/d of nicotine bitartrate. This dose produces plasma cotinine (a metabolic by-product of nicotine) levels in the pups ranging from 60 to 92 ng/ml , which iThese studies were designed to test the hypothesis that DNE is associated with decreased drive to the tongue muscles in response to airway occlusion . Neonatal rat pups ranging in age from P3–P7 were lightly anesthetized with a mixture of ketamine (30 mg/ml), xylazine (6 mg/ml), and acepromazine (3 mg/ml), injected subcutaneously at a volume corresponding to ∼0.35 µl/g body weight. Pain sensitivity was checked via multiple paw pinches initiated 10 min after the time of injection. Supplemental anesthetic was added until paw retraction on pinching was abolished. Whole muscle electromyographic (EMG) activity of the genioglossus muscle  was recorded using fine wire electrodes inserted into the area underneath the tip of the mandible . An addiAfter implantation of the EMG electrodes, the animal was inserted into a 32 ml head-out plethysmograph in the supine position A. A neckPost hoc analysis was by Tukey’s test. The difference in EMG onset latency was tested with the unpaired t test; p < 0.05 was taken as the threshold for statistical significance ; the remainder were not used in the main analysis. However, we did find that 12 of the 135 animals failed to recover from nasal occlusion, and as shown in In the course of working out the These experiments were designed to examine the influence of DNE on AMPA-mediated glutamatergic synaptic input to XIIMNs. As indicated below, we studied both spontaneous EPSCs (sEPSCs) and miniature EPSCs (mEPSCs) inputs. This dual focus is important, as sEPSCs reflect both action potential-mediated glutamate release, as well as inputs due to the random, quantal release of glutamate from presynaptic terminals. Moreover, nAChRs are located presynaptically on the soma, dendrites and axon end-terminals of glutamatergic neurons as well as postsynaptically on XIIMNs, and exposure to nicotine could alter nAChRs in all of these locations. Therefore, recording both miniature and spontaneous events can help determine whether the actions of DNE on XIIMNs are presynaptic or postsynaptic.2/5% CO2) artificial cerebrospinal fluid (aCSF), composed of the following: 120 mM NaCl, 26 mM NaHCO3, 30 mM glucose, 1 mM MgSO4, 3 mM KCl, 1.25 mM NaH2PO4, and 1.2 mM CaCl2 with pH adjusted to 7.4 and osmolarity to 300–325 mOsm. The brainstem was extracted and glued to an agar block, rostral surface up, and two to three transverse medullary slices (300–300 microns) containing the hypoglossal motor nucleus were cut in a vibratome  filled with ice-cold aCSF. The slices were then transferred to an equilibration chamber containing fresh, oxygenated, room temperature aCSF and allowed to recover for 1.5 h before recording.Pups of either sex were removed from their cages, weighed, anesthetized on ice and decerebrated at the coronal suture. The vertebral column and ribcage were exposed and placed in cold (4–8°C) oxygenated (95% O2/5% CO2) aCSF at a rate of 1.5–2 ml/min. XIIMNs were visualized with an Olympus BX-50WI fixed-stage microscope  with differential contrast optics and a video camera . Recordings were made with glass pipettes (tip resistance 3–7 MΩ) pulled from thick-walled borosilicate glass capillary tubes . We used a CsCl based intracellular solution containing the following: 130 mM CsCl, 5 mM NaCl, 2 mM MgCl2, 1 mM CaCl, 10 mM HEPES, 2 mM ATP-Mg, 2 mM aucrose, with pH adjusted to 7.2 and osmolarity of 250–275 mOsm. Under these conditions, the chloride reversal potential is ∼0 mV  and therefore both EPSCs and IPSCs are inward at a holding potential of –75 mV. Filled pipettes were attached to a head stage mounted in a micromanipulator . The head stage was connected to a Multiclamp 700B amplifier, and the signals were digitized with a Digidata 1440A A/D converter (Molecular Devices).Equilibrated slices were transferred to a recording chamber maintained at 27°C  and perfused with oxygenated -2-amino-5-phosphonopentanoic acid (AP-5), strychnine hydrochloride, and bicuculline methiodide to antagonize the NMDA receptors the glycine receptors, and the GABAectively . In the A and glycine receptors, as above, to isolate AMPA-mediated excitatory events. This cocktail was superfused for 3 min, followed by the addition of tetrodotoxin (TTX) for 2 min to block action potential firing  and TTX . All drugs were mixed in aCSF on the day of the experiment from previously mixed aliquots that were frozen and stored at 0–2°C. Antagonists were used at concentrations known to be effective based on our previous studies or the literature. Nicotine was used at the highest dose that produced presynaptic effects (increased frequency of sEPSCs in pilot experiments) without producing a significant inward current, which we found to be 0.5 μM. This is important, as pilot studies showed that higher concentrations of nicotine  activates postsynaptic nAChRs and evokes an inward current, that decreases the ability to discriminate sEPSCs/mEPSCs. AMPA was used at a concentration that produced an approximately half maximal response (2.5 μM), based on dose-response experiments previously performed in our lab. The drug solutions were oxygenated and maintained at 27°C and perfused into the recording chamber at a rate of 1.5–2 ml/min.Data from a total of 72 cells are reported, with 36 cells from DNE neonates and 36 cells from control neonates. Cell numbers for each experiment are summarized in -3, 30 mM glucose, 1 mM MgSO4, 3 mM KCl, 1.25 mM NaH2PO4, and 1.2 mM CaCl, gassed with 95% O2/5% CO2, with pH adjusted to 7.45–7.5. Force was measured with a transducer (Kent Scientific) attached to the central tendon of the hemidiaphragm, and the phrenic nerve was drawn into a suction electrode, which was referenced to a bath ground and connected to a stimulator (Grass S88). Two silver/silver-chloride discs were pinned to the bath on either side of the muscle strip and connected to a second channel on the stimulator for direct depolarization of the muscle fibers, bypassing the neuromuscular junction. Force and the output of the stimulator were digitized and monitored on a computer using Spike II hardware and software (CED).To estimate the influence of DNE on the integrity of the neuromuscular junction, and as a substitute for the hypoglossal nerve-tongue neuromuscular junction (see Discussion), we used neonatal rat (P1–P5) phrenic nerve-hemidiaphragm preparations. Diaphragm muscle was excised and secured in a dish perfused with warmed (37°C), oxygenated Krebs solution, containing the following: 123 mM NaCl, 26 mM NaHCOTo measure muscle twitch force and contraction speed in response to direct muscle stimulation we delivered single, 0.2-ms supramaximal pulses to the bath electrodes. We also measured the decline in force following 5 min of intermittent, supramaximal direct muscle stimulation, while in another set of animals we stimulated the phrenic nerve. Both muscle and nerve were stimulated with 0.2-ms pulses, delivered in 330-ms trains at 40 Hz, with a train delivered every 2 s. In additional sets of animals, we estimated the contribution of neuromuscular transmission to force loss by applying stimulus trains to the phrenic nerve, as above, while superimposing direct muscle stimulation every 15 s. Neuromuscular transmission failure was computed by comparing the force loss during nerve stimulation with that evoked by direct muscle stimulation C, as desin vivo experiments, changes in EMG activity during nasal occlusion were analyzed with two-way ANOVA, with time and treatment the main factors. Post hoc analysis was by Tukey’s test. The difference in EMG onset latency was tested with the unpaired t test; p < 0.05 was taken as the threshold for statistical significance  test using SPSS (IBM).We analyzed the IEI and amplitude for both sEPSCs and mEPSCs in two ways . First, post hoc analyses using the Holm–Sidak multiple comparisons test, with the alpha level set at p < 0.05. We used an unpaired t test to compare the mean peak current in control and nicotine exposed cells , age (P1–P2 vs P3–P5) and experimental condition . When the ANOVA was significant, we conducted ed cells . We noteed cells . This ist test. As above, p < 0.05 was the threshold for statistical significance , as shown in the representative recordings in We began our study by assessing the impact of DNE on neural drive to the GG muscle of the tongue, which is innervated by XIIMNs and plays a major role in keeping the upper airway open . We did 2 analysis , with an adjusted odds ratio of 3.5.As indicated in Materials and Methods, we did the nasal occlusion test in 135 neonates and noted that 12 of the 135 never recovered, and instead entered a state characterized by terminal gasping. The pattern of breathing in animals that did not recover most often approximated that shown in The age and weight of the animals used in these experiments are shown in To evaluate the influence of DNE on network level AMPA receptor-mediated synaptic events under baseline conditions, we evaluated differences in the IEIs and the amplitude of sEPSCs in control and DNE cells. The upper traces in p = 0.049, based on 675 and 749 events at baseline and with acute nicotine challenge, respectively) and DNE cells . In animals aged P3–P5, nicotine challenge again shifted the cumulative probability distributions to the left in control animals  and DNE cells . In animals aged P3–P5, nicotine challenge again shifted the cumulative probability distributions to the left in control animals  including peak twitch tension , time to peak tension , and ½ relaxation time  were the same in diaphragm strips from control and DNE animals. We note that our values for each of these variables are similar to those reported in 7-d-old neonatal rats . Figure 2 and a decrease in O2, leading to an increase in the release of several neurotransmitters, including ACh  is accompanied by an increase in blood COding ACh . The inco XIIMNs , these oo XIIMNs . Below, in vitro techniques to evaluate how DNE alters neurotransmission and the development of hypoglossal motoneurons, which innervate the tongue muscles. In contrast, we are unaware of any studies on the influence of DNE on the control of tongue muscle activity in vivo. Here, we show that DNE blunts the magnitude and delays the activation of the GG EMG during imposed nasal occlusions. Although we are unable to establish the mechanisms underlying these observations, there are clues from previous work. First, nasal occlusion prevents lung inflation, and the absence of inflation releases hypoglossal motoneurons from inhibition, leading to increased GG activity, at least in adult rodents ; (2) a critical developmental period; and (3) an exogenous stressor. Maternal smoking is indeed the number one risk factor for SIDS and apparent life threating events in neonates, but the lack of a suitable animal model to probe the mechanisms underlying this association has been elusive. It is noteworthy that SIDS risk is highest in infants aged four to five months, while it has been suggested the first two weeks of life in the rat corresponds to 28–40 weeks of gestation in humans  and apparent life-threatening events (ALTEs) is the triple risk hypothesis , which in humans , so alth