Bidens pilosa, Galinsoga ciliata, and Eryngium foetidum against 6 clinical strains of Helicobacter pylori in vitro and in vivo. Broth microdilution method was used in vitro. In vivo, Swiss mice were inoculated with H. pylori and divided into 5 groups; the control group received the vehicle and the four others received 125, 250, and 500 mg/kg of methanol extract of Eryngium foetidum and ciprofloxacin (500 mg/kg) for 7 days, respectively. Helicobacter pylori colonization and number of colonies in gastric biopsies culture were assessed on days 1 and 7 after treatment. The lowest MIC value (64 μg/mL) and the best spectrum of bactericidal effect (MBC/MIC = 1) were obtained with the methanol extract of Eryngium foetidum. The number of H. pylori infected animals was 17% (plant-extract) and 0% (ciprofloxacin) compared to 100% for the infected untreated group. Plant-extract (381.9 ± 239.5 CFU) and ciprofloxacin (248 ± 153.2 CFU) significantly reduced bacterial load in gastric mucosa compared to untreated, inoculated mice (14350 ± 690 CFU). Conclusion. The present data provided evidence that methanol extract of Eryngium foetidum could be a rich source of metabolites with antimicrobial activity to fight Helicobacter pylori infections.This study was performed to evaluate the antimicrobial activities of extracts of Helicobacter pylori is a Gram negative microaerophilic helical bacillus that affects the gastric mucosa and can be found attached to epithelial cells of the human stomach [ H. pylori develop related gastroduodenal disorders during their lifetime [ Helicobacter pylori positive around the world, with the developing countries having a prevalence of 80%–90% and industrialized countries like the United States having ≈35%–40% [ H. pylori infection is high in developing countries (≈4%–15%) compared with industrialized countries (0.5%) [ H. pylori infection. This eradication therapy entails two of the following antimicrobial agents; metronidazole, amoxicillin, tetracycline, or clarithromycin in association with a proton pump inhibitor or bismuth [ H. pylori strain is the most common causes of treatment failure [ in vitro assessment of anti-Helicobacter properties of medicinal plant products [ in vitro anti-Helicobacter properties become ineffective in vivo [ in vitro therapeutic activity of plant products must be confirmed by an in vivo test. Even though no animal model of the H. pylori infection exactly matches the human disease, experimental models on some animals such as primates, domestic cats and dogs, gnotobiotic piglets, ferrets, Mongolian gerbils, and mice provide important insights into the in vivo evaluation of new antimicrobials. stomach . It is a stomach . Approxilifetime . Approxis 0.5%) . The rec% . The r failure . The fai failure . Consequproducts . Howeverries ≈4%–% compare in vivo . TherefoEryngium foetidum is an annual small plant (approximately 30 cm high) in the family Apiaceae, used as a medicinal herb and spice. It is used as health foods in view of its significant amount of calcium, iron, carotene, riboflavin, proteins, and vitamins A, B, and C content [ content . In trad content , 20. In  content . Studies content –25. H. pylori which is a major cause of gastric ulcer has not been experimentally confirmed [ in vitro and in vivo antibacterial activity of Eryngium foetidum on clinical strains of H. pylori in order to identify potential sources of materials for the synthesis of new drugs to fight Helicobacter pylori infections.Despite its popular uses to calm stomach pain, its activity againstonfirmed . This st H. pylori were freshly isolated and suspended in Brain Heart Infusion (BHI) broth supplemented with 5% horse serum and 20% glycerol and stored at −80°C until further use.The clinical microorganisms used here were obtained from the Gastroenterology Department of Laquintinie Hospital, Douala, Cameroon. These microorganisms were collected from gastric biopsies of patients complaining of upper gastrointestinal pain, hematemesis, or/and dyspepsia requiring gastroendoscopic examination. Six strains ofSwiss albino mice of 18–22 g weight obtained from Animal House, University of Dschang, Cameroon, were selected as experimental animals. These animals were fed with standard pellet diet and tap water.Throughout the experiments, all animals received human care according to the criteria outlined in the internationally accepted principle guidelines of the European Union on Animal Care (CEE Council 86/609). Animal experiments were designated and approved by the Cameroon National Ethics Committee (Reg. number. FWA-IRB00001954). Bidens pilosa (Asteraceae) and Galinsoga ciliata (Asteraceae) were collected in Dschang (west region of Cameroon) and leaves of Eryngium foetidum (Apiaceaes) in Kumba (southwest region of Cameroon). Identification of the plant was done at the National Herbarium Yaoundé . The plant materials were then air-dried at room temperature and ground into a fine powder.The leaves ofGen gas pack were obtained from Oxoid, Basingstoke, England. Doxycycline , metronidazole , and ciprofloxacin  were used as reference antibiotics for the determination of the MIC and MBC and for the in vivo test were obtained from local pharmacy. P-Iodonitrotetrazolium chloride  was used to indicate microbial growth [Culture media , lacked horse blood, Horse Serum, Vitox Supplement) and Campyl growth .Dried powdered plant material was weighed (250 g) and soaked in 3.5 L of ethyl acetate (EA) or methanol (MeOH) for 72 hours. The plant mixture was then filtered and concentrated under reduced pressure using a rotary evaporator. The extract was further concentrated by allowing it to stand overnight in an oven at 30°C.H. pylori α1, H. pylori α2, H. pylori α3, H. pylori α4, H. pylori α5, and H. pylori α6), identified using Gram staining and enzymatic properties , were employed to evaluate the in vitro anti-Helicobacter activity of the plant-extracts. Methanol and ethyl acetate extracts of Bidens pilosa, Galinsoga ciliata, and Eryngium foetidum were used for the determination of MICs by the INT broth microdilution method [μL of the extract-containing culture medium. Control wells were prepared with culture medium and bacterial suspension and broth, only respectively. Ciprofloxacin, doxycycline, and metronidazole at concentration ranges of 0.002 to 0.128 mg/mL were used as positive control. The plates were covered with a sterile plate sealer; the contents of the wells were mixed with a shaker and incubated for 3 days at 37°C under microaerophilic conditions. After incubation, 40 μL of 0.2 mg/mL INT was added per well and incubated at 37°C for 30 min. Living bacteria reduced the yellow dye to pink. The sample concentration that prevented the color change of the medium and exhibited complete inhibition of microbial growth is known as the MIC. The MBC was determined by adding 50 μL aliquots of the preparations which prevented the color change of the medium after incubation during MIC assays, to 150 μL of BHI-serum. These preparations were incubated at 37°C for 72 h under microaerophilic conditions. The lowest concentration of extract, which did not produce a color change after addition of INT, was considered as the MBC. Each MIC and each MBC were determined in triplicate and the mean values are recorded.The clinical strains .Methanol extract of Eryngium foetidum extract, once daily for seven consecutive days. The mice were allowed to fast for 12 h after the last day of treatment and six from each group were sacrificed with ethyl ether. The rest of animal were sacrificed after a posttreatment period of seven days. The stomach of each animal was removed and opened through the longer curvature with sterile surgical instruments. The stomach and duodena, covering all subtypes of mucosa, were used for culturing H. pylori.Inoculated mice were distributed into five groups of 12 animals and allowed to rest for 24 h after the last inoculation. Group I, a negative control group, received sterile solution of the vehicle; group II was treated with ciprofloxacin (500 mg/kg) and served as positive control; test groups III to V were treated, respectively, with 125, 250 and 500 mg/kg of H. pylori was identified through macroscopic characteristics , microscopic characteristics and Gram staining (Gram negative curved rods), and enzymatic properties . The bacterial load was estimated by colony count and expressed as log10 CFU per milliliter of homogenate.Stomach biopsies were scraped off and homogenized with 0.5 mL of PBS in a 1.5 mL Eppendorf tube. The homogenized sample was serially diluted 10-fold. Each 0.1 mL of homogenate was plated on supplemented Columbia Agar and incubated at 37°C for 2 to 3 days under microaerophilic conditions . H. pylo10. The analysis of variance followed by the paired Student's t-test was used for the statistical evaluation of data. The differences between groups were considered significant at p < 0.05.The results were expressed as means ± standard deviation. Before analysis, bacterial densities were expressed in logHelicobacter activity with each other with MIC values ranging from 64 to >1024 μg/mL. The methanol extract of E. foetidum shows the best activity for MIC values between 64 and 512 μg/mL against 3/6 (50%) evaluated strains. Methanol extract from E. foetidum also displayed the best spectrum of bactericidal effect with a ratio of MBC/MIC 1 obtained on two tested H. pylori strains. MIC values from 128 to 512 μg/mL were also recorded with E. foetidum (AE), Bidens pilosa (MeOH), Bidens pilosa (AE), Galinsoga ciliate (MeOH), and Galinsoga ciliata (AE) extracts, respectively, against 2/6 (33.33%), 4/6 (66.66%), 1/6 (16.66%), 4/6 (66.66%), and 4/6 (66.66%) of the evaluated bacteria. The best MIC value (64 μg/mL) was obtained with the methanol extract from E. foetidum against H. pylori α1 and H. pylori α2.Plant species showed different anti- E. foetidum.According to  H. pylori positive animals in treated groups (plant-extract and ciprofloxacin) was lower than the number recorded in the untreated infected group (control group)  . None of H. pylori colonization of gastric mucosa as shown in the number of bacterial counts ( E. foetidum (381.9 ± 239.5 CFU) and ciprofloxacin (248 ± 153.2 CFU) compared to that recorded with untreated infected mice (14350 ± 690 CFU).Infected mice showed stablel counts . The bac H. pylori strains. In vitro, the tested plants displayed selective antibacterial activities and this activity was different within the same plant species from one extraction solvent to another. In fact, the type of solvent used largely affects the effectiveness of the extracts. The differences observed in the antibacterial activities of the plants species could be due to the differences in their chemical composition and in the mechanism of action of their bioactive constituents [μg/mL, moderate when being between 100 and 625 μg/mL, and weak when being greater than 625 μg/mL [μg/mL obtained with E. foetidum methanol extract against H. pylori α1 and H. pylori α2 can be considered significant. The active principles of the extract of this plant should be assignable to different secondary metabolites belonging to a wide variety of compound classes. The preliminary phytochemical analysis of the tested plant-extract revealed the presence of alkaloids, phenols, flavonoids, anthraquinones, and steroid  and ciprofloxacin significantly reduced H. pylori colonization of gastric mucosa in infected mice as compared to infected untreated mice. Thus, our data showed that the anti-Helicobacter activity of the methanol extract of E. foetidum was effective in vitro and in vivo. The activity of the plant-extract demonstrated herein is very important when considering that the tested plant-extracts are from edible plant parts and also when considering the medical importance of the tested bacteria. In fact, H. pylori is a virulent pathogen as evidenced by its low infective dose and high prevalence in human populations and the principal cause of type B gastritis, peptic ulcer disease, gastric adenocarcinoma, and MALT-lymphoma [ H. pylori drastically reduced the limited range of antibiotics that have efficacy in the treatment of this infection leading globally to an increase in the frequency of therapeutic failure [Since thelymphoma . It has lymphoma . Further failure . E. foetidum could be a rich source of metabolites with antimicrobial activity to fight Helicobacter pylori infections. Further investigations on purification and structure elucidation of the compounds are in progress.In conclusion, the present data provided evidence that methanol extract of


Malnutrition is a global public health problem especially in developing countries experiencing war/conflicts. War might be one of the socio-political factors influencing malnutrition in Sub-Saharan African (SSA) countries. This study aims at determining the influence of war on corruption, population (POP), number of population malnourished (NPU), food security and life expectancy (LE) in war-torn SSA countries (WTSSA) by comparing their malnutrition indicators.T test, multivariate and Pearson correlation analyses were performed to determine the relationship between CPI, POP, GHI, FSI, NPU, male LE (MLE) and female LE (FLE) in WTSSA at p < .05. Data were presented in tables, means, standard deviation and percentages.Fourteen countries in WTSSA were stratified into zones according to war incidences. Countries’ secondary data on population (POP), NPU, Food Security Index (FSI), corruption perceptions index (CPI), Global Hunger Index (GHI) and LE were obtained from global published data. Mean NPU, CPI, GHI, POP, FSI, MLE and FLE in WTSSA were 5.0 million, 28.3%, 18.2%, 33.8 million, 30.8%, 54.7 years and 57.1 years, respectively. GHI significantly influenced LE in both male and female POP in WTSSA. NPU, CPI, FSI, GHI and FLE were not significantly different according to zones except in MLE.Malnutrition indicators were similarly affected in WTSSA. Hunger influenced life expectancy. Policies promoting good governance, equity, peaceful co-existence, respect for human right and adequate food supply will aid malnutrition eradication and prevent war occurrences in Sub-Saharan African countries. Malnutrition is a global public health problem in Sub-Saharan Africa (SSA) –3. One oWar and conflict are rampant in many developing countries especially in SSA due to conflicts over basic resources such as food and water, access and control over rich minerals, unresolved political issues and agendas, effect of global financial crisis, rising costs of living, human rights violations, land and border disputes, harsh dictatorship, high unemployment especially among educated youths, frustration from decades of living under authoritarian and corrupt leaders and environmental degradation as reported by Goldson  and ShahOne of the most significant consequences of war is the number of refugees. There have been over nine million refugees and internally displaced people from conflicts in Sub-Saharan Africa . During Few studies have compared the influence of war on malnutrition indicators such as food security, life expectancy, number of population undernourished and hidden hunger in developing countries especially those experiencing one form of war or conflict to ascertain the actual relationship between these indicators. This study aims at bridging this gap.Only countries experiencing war/conflict in SSA were selected. Countries were excluded if they do not have complete relevant data  available. Global Food Security Index (GFSI) 2013 and 2014 only had records for 107 and 109 countries, respectively. Many developing countries do not have GFSI and GHI data. Only 14 out of 26 countries experiencing war or conflicts in SSA that had complete relevant data available were included.The researcher surfed the net to identify ‘SSA countries undergoing war or some form of conflicts’. Stratified sampling technique was used in selecting countries based on incidence of war. Sub-Saharan African countries experiencing war with their socio-economic and malnutrition indices were grouped into zones: West Africa, East Africa, Central Africa and North Africa. Out of 26 SSA countries experiencing war, only 14 with complete relevant data were purposely selected. Qualitative and quantitative data were collected. Secondary data on countries’ corruption perceptions index (CPI), Global Food Security Index (GFSI), population (POP), Global Hunger Index (GHI), number of population undernourished (NPU) and life expectancy (LE)  scores were collected from published indexes –14.T test, multivariate regression (Wilks’ lambda) and Pearson correlation analyses were done to determine the relationship between GHI, CPI, GFSI, POP, NPU and LE in war-torn countries in SSA in zones. Data were presented in tables, means, standard deviation and percentages.Data was collected and captured in an Excel sheet. p > .05). But GHI influenced life expectancy in both male and female populations (p < .05). Also, FLE was significantly different from MLE (p < .05). Table p < .05). War similarly affected all malnutrition indicators in WTSSA as shown in Table Table p < .05) as shown in Table Only 53.9% of countries in developing countries experiencing some kind of war or conflicts had complete data. This is an indication that war periods precipitate loss of data which could aid policy decisions. Countries in West Africa experienced the highest incidence of war (50.0%) followed by East Africa 28.6%), Central Africa 14.3%) and then North Africa 7.1%). The high number of population malnourished (5.0 million) in war-torn countries of SSA justifies the low GHI (18.2%) and GFSI (30.8%) as shown in Table 4.3% and %, Centra.1%. The Chronic hunger results in severe malnutrition which shortens the length of time people could live. Significant difference existed in the MLE across the zones. Central Africa had the least MLE (49.5 years) followed by the Western zone (52.7 years). These zones experience war and conflicts more than the East zone. East Africa had a better mean corruption perception index 32%) even though it is still low, the highest GFSI (34%), MLE (59%) and FLE (62%) (Table 2% even tThe East zone had the highest number of people under nourishment (NPU 6.6 million) and was the second zone with a higher Global Hunger Index (18.9%), yet the zone scored highest in MLE and FLE indicating that probably in war situations, other factors could be employed to help the citizens to live better healthy lives such as adequate medical care, food supplementation, nutrition education, relief materials from other countries and food diversity using local staples. It is normally during war that new nutritious staples are discovered. East Africa might have had alternative sources of food during the war which made its GFSI the highest among the zones in SSA (33.8%). The significant difference between FLE and MLE might be because males are recruited into war at young ages.All the mean values for malnutrition indicators in WTSSA were generally low, below 50%. Malnutrition indicators were similarly affected in WTSSA especially male and female life expectancies. Policies promoting good governance, equity, peaceful coexistence, respect for human right and adequate food supply will help to eradicate malnutrition and prevent war occurrences in Sub-Saharan African countries.


Purpose. This study was to investigate the effects of cornel iridoid glycoside (CIG) on spinal cord injury (SCI) in rats. Methods. The thoracic cord (at T9) of rats was injured by clip compression for 30 sec. Locomotor function was assessed using the Basso, Beattie, and Bresnahan (BBB) rating scale. Neuroanatomic stereological parameters as well as Nogo-A, p75 neurotrophin receptor (p75NTR), and ROCKII expression were measured by histological processing, immunohistochemistry, and stereological analyses. The axons passing through the lesion site were detected by BDA tracing. Results. Intragastric administration of CIG (60 and 180 mg/kg) improved the locomotor impairment at 10, 17, 24, and 31 days post-injury (dpi) compared with untreated SCI model rats. CIG treatment decreased the volume of the lesion epicenter (LEp) and increased the volume of spared tissue and the number of surviving neurons in the injured spinal cord at 31 dpi. CIG promoted the growth of BDA-positive axons and their passage through the lesion site and decreased the expression of Nogo-A, p75NTR, and ROCKII both in and around the LEp. Conclusion. CIG improved the locomotor impairment, decreased tissue damage, and downregulated the myelin-associated inhibition signaling pathway in SCI rats. The results suggest that CIG may be beneficial for SCI therapy. Spinal cord injury (SCI) is a severe, often life-threatening, and debilitating clinical condition, affecting mainly young people, and the treatment incurs massive costs associated with ongoing care. This makes SCI a serious social and economic problem , 2. The SCI pathogenesis also involves the myelin-associated inhibition signaling pathway that plays an important role in restricting axonal growth and neurological recovery . Nogo-A Cornus officinalis is a member of the Cornaceae family and was first recorded in Shennong's Classic of Materia Medica about 2000 years ago in China. In traditional Chinese medicine, Cornus officinalis is used to tonify the liver and the kidney for the treatment of vertigo, aching back, spontaneous emission, and sweating [ Cornus officinalis is also used to treat stroke in combination with other herbs [ Cornus officinalis. In the previous studies, we found that CIG inhibited inflammation and apoptosis, promoted neurogenesis and angiogenesis in the brain, and improved neurological deficits after focal cerebral ischemia in rats [sweating . Cornus er herbs . Cornel  in rats , 18. We  in rats . HoweverIn the present study, we used a clip compression method to establish the SCI rat model, which is considered to be a suitable model for SCI studies and related therapeutic interventions , 21. We  Cornus officinalis, which was purchased from Tong Ren Tang Company, Beijing, China. The technique of extraction was developed by our laboratory and described briefly in a previous paper by Yao et al. [Cornel iridoid glycoside (CIG) was extracted from the sarcocarp ofo et al. . The purAdult female Wistar rats  were obtained from Beijing Vitalriver Experimental Animal Co. . They were housed in a controlled environment with a temperature at 24 ± 1°C, a 12/12 h dark/light cycle, with the humidity maintained at 60% to 70%. During the whole experiment, rats were free to get food and water. All experiments were done according to the rules of the Provisions and General Recommendations of Chinese Experimental Animal Administration Legislation.n = 12 in each group): the sham control group, the SCI model group, SCI+CIG 60 mg/kg group, and SCI+CIG 180 mg/kg group. CIG was dissolved in normal saline and administered intragastrically starting at 4 h after the surgery of SCI and once a day for 31 days. The sham control and the model groups received an equal volume of normal saline.Rats were randomly divided into 4 groups  set on a syringe pump . The syringe remained for an additional 5 min before retraction as described before [At 10 days post-injury (dpi), 3 rats in each group were selected randomly for biotinylated dextran amine (BDA) tracing surgery. The animals were anesthetized and positioned on a stereotaxic apparatus. A dental drill was used to make burr holes through the cranium bilaterally overlying the sensorimotor cortex   and at td before . A thin The behavioral assessment was performed weekly at 3, 10, 17, 24, and 31 dpi, respectively. Before the surgery, the animals were trained over a period of 5 days for acquaintance with the environment and technicians as preparation for the behavioral test. The open-field locomotor test was conducted by using the 21-point Basso, Beattie, and Bresnahan (BBB) locomotor rating scale . The ratμm thick, and horizontal sections in a cryostat for the stereological histological analyses, and then two sections from every eighth section were directly mounted on gelatin-coated slices for myelin basic protein (MBP) and immunohistochemistry staining.For the histological analysis of the spinal cord, all rats were anesthetized with 10% chloral hydrate  and perfused transcardially with 0.01 M PBS (pH 7.4). The spinal cords were removed and postfixed in 4% paraformaldehyde in 0.01 M PBS for 72 h and then switched to 30% sucrose/0.1 M PBS. The segment was selected for analysis from rostral to caudal areas in which the lesion epicenter was on center and throughout the entire injury . All the segments were moved and embedded in optimum cutting temperature compound at −20°C. After being frozen in isopentane, each segment was cut into serial, 30 In order to measure the stereological parameters of spinal cord lesion, we used MBP for presentation of myelin and counterstained with cresyl violet for Nissl substance , 28. Bef2O2/methanol for 15 min and then exposed overnight to the primary antibodies including anti-Nogo-A , anti-p75NTR , and anti-ROCKII , respectively. The sections were incubated with goat anti-rabbit biotinylated IgG  for 1 h at room temperature. After being incubated with HRP streptavidin , immune complexes were visualized by incubation of sections in the DAB system. The sections were then coverslipped with neutral balsam as described above.For the detection of Nogo-A, p75NTR, and ROCKII, randomly chosen slide series mounted with evenly spaced tissue sections were processed for immunohistochemical staining. The sections were incubated in 3% Hμm thick, parasagittal sections in the same cryostat for BDA tracing. BDA was visualized using the Neuro Trace™ BDA Neuronal Tracer Kit . Sections were rinsed several times in 0.01 M PBS and then incubated in the avidin-HRP working solution (0.2 μg/ml) overnight at 4°C. The next day, the tissue sections were rinsed in PBS three times for 5 min each. DAB working solution (0.1% concentration with 0.03% H2O2) was applied to the sections under light microscope observation. After being terminated in distilled water, the sections then were air-dried, dehydrated, defatted, and coverslipped. An Olympus microscope  was used for photomicrography every third BDA-labeled sections intact in a bright field with the assistance of Volocity 6.0 software  at 40x magnification. The BDA-labeled axons at the rostral and the caudal area within 1 mm from the edge of lesion epicenter (LEp) were counted and summed up as they were longer than 40 μm [PBDA) was calculated as PBDA =  × 100%.Each segment from the BDA-labeled part was cut into serial, 25 an 40 μm . The perA Leica microscope  with motorized stage and StereoInvestigator  were used in stereological analysis. According to rules of the unbiased random sampling , startinVLEp) and spared tissue  were measured from MBP immunostained sections using the Cavalieri estimator probe in StereoInvestigator [R) of VLEp, VGM, and VWM to the volume of the total segment (VT), which were computed using the following expression to minimize the influence of tissue processing and staining methods: RX = (VX/VT) × 100%.The volumes of lesion epicenter  stained with cresyl violet in entire grey matter of spared tissue was quantified by the Optical Fractionator probe [μm × 100 μm, and the space between the adjacent frames was 400 μm. In order to distinguish neurons from glial cells in the quantitative analysis, only neurons with an ovoid, spherical, triangular, or multipolar profile and the soma diameter larger than 10 μm with an intact cellular membrane and a clearly defined nucleus were counted [NRN in the spare tissue was performed as previously described [DRN, cells per mm3) was calculated in each group as DRN = (NRN/VSP).The number of residual neurons  over time for 4 groups were analyzed using repeated-measures ANOVA with the between-groups factor, followed by Fisher's least significant difference (LSD) post hoc test. All stereological quantifications and PBDA were analyzed using one-way ANOVA to compare differences among the four groups followed by Fisher LSD post hoc analysis. The significance was set at P < 0.05.All data were expressed as mean ± standard error (mean ± SEM) and analyzed using the software package SPSS 11.0 . Changes in BBB scores (SBBB) in the SCI model rats decreased significantly compared with those in sham-operated animals at all time points (P < 0.01). The intragastric administration of CIG at a dose of 60 mg/kg once a day starting at 4 h after the SCI surgery significantly elevated SBBB at 24 and 31 dpi (P < 0.05). In the CIG 180 mg/kg-treated animals, SBBB was significantly increased at 10, 17, 24, and 31 dpi  when compared with the SCI model rats.The rat locomotor function was evaluated by using the Basso, Beattie, and Bresnahan (BBB) locomotor rating scale in the open-field test. SBBB interpretation [SBBB ≤ 7, the rat is likely to never walk with its hindlimbs again, which means the rat is completely uncured. By contrast, SBBB ≥ 10 predicts that the rat has reached a level of recovery sufficient to walk. If SBBB is between 7 and 10, the rat has a chance to eventually walk. Therefore, we calculated the percentages of rats in these three categories in all injured groups at 31 dpi. SBBB ≤ 7 and no rat got SBBB ≥ 10 in the SCI model group. However, in the CIG 60 mg/kg-treated group, 33.3% of the rats obtained SBBB ≥ 10, 55.6% had SBBB between 7 and 10, and only 11.1% presented SBBB ≤ 7. In the CIG 180 mg/kg-treated group, 55.6% of the rats obtained SBBB ≥ 10, and the others presented SBBB between 7 and 10. The results demonstrated that CIG dose-dependently improved the locomotor impairment and promoted the functional recovery in SCI rats.According to the retation , if SBBVT), which meant that the tissue preparation procedure was consistent and the comparison of the other stereological parameters was on the same baseline. In the SCI model rats, the volume of the lesion epicenter (VLEp) significantly increased (P < 0.01), and the volume of the spared grey matter (VGM) and white matter (VWM) obviously decreased (P < 0.05). The treatment with 60 and 180 mg/kg CIG markedly reduced VLEp  and increased the spared VWM (P < 0.05) compared with the SCI model rats.In order to measure the SCI stereological parameters, myelin basic protein (MBP) immunostaining with cresyl violet counterstaining was used in this experiment. The representative coronary section of the injured spinal cord in the different groups is presented in VLEp, VGM, and VWM) to the volume of total segment (VT). VLEp to VT (RLEp = VLEp/VT) significantly increased (P < 0.01), and the ratios of VGM and VWM to VT (RGM = VGM/VT and RWM = VWM/VT) markedly decreased (P < 0.01) in the SCI model rats compared with those in the sham control animals. The treatment with 60 and 180 mg/kg CIG significantly reduced RLEp  and elevated RWM  in SCI rats; and the 180 mg/kg CIG treatment also obviously increased RGM (P < 0.05). The results in To minimize the systematic errors in the tissue preparation procedure, we calculated the ratio of the volume of different parts (NRN) in the spared tissue (GM and WM) of the injured spinal cord is presented in NRN significantly decreased in the SCI model rats compared with the sham-operated control rats (P < 0.01). CIG treatment dose-dependently increased NRN, in which there was a significant difference between the CIG 180 mg/kg-treated animals and the SCI model rats (P < 0.01).The residual neurons measured by cresyl violet staining for Nissl substance are displayed in DRN = NRN/VSP, in which VSP represents the volume of spared tissue, including GM and WM). DRN was found in the SCI model rats (P < 0.01), and the 60 and 180 mg/kg CIG treatments obviously increased DRN (P < 0.01) in SCI rats. The results in For the purpose of minimizing the systematic error in the tissue preparation procedure, we calculated the average density of residual neurons (PBDA) significantly decreased in the SCI model group compared with that in the sham-operated rats (P < 0.01). The treatments with 60 and 180 mg/kg CIG obviously increased PBDA in SCI rats . These results suggest that CIG may promote axon growth and passage through the LEp, implying the functional recovery of axonal transport after SCI.In order to investigate the axon growth and passage through the LEp in the spinal cord, BDA was injected into the sensorimotor cortex and the red nucleus at 10 dpi and then transmitted through the corticospinal tract (CST) and rubrospinal tract (RST). P < 0.05, P < 0.01); and CIG (60 mg/kg) also declined this ratio for p75NTR (P < 0.05). Around the LEp, CIG treatment (60 and 180 mg/kg) obviously reduced the number of Nogo-A, p75NTR, and ROCKII positive cells in a range of 1–3 mm beyond the outmost edge of LEp, respectively . These results demonstrated that CIG treatment inhibited the expression of Nogo-A, p75NTR, and ROCKII both in the LEp and around the LEp of the injured spinal cord, suggesting a downregulation effect on the myelin-associated inhibition signaling pathway.The expression of Nogo-A, p75NTR, and ROCKII in the myelin-associated inhibition signaling pathway was measured by immunohistochemical and stereological methods. The present study indicated that CIG treatment improved the locomotor impairment, decreased the LEp volume, preserved more spared tissue and residual neurons, promoted more biofunctional axons passing through the LEp, and reduced Nogo-A, p75NTR, and ROCKII expression in the injured spinal cords of rats.The BBB scale is considered to be an effective and predictive measure to recognize behavioral outcomes due to different injuries and to predict anatomical alterations at the lesion site . It has The stereological approach provides an objective unbiased assessment of the structural changes in biological systems and it has been used to analyze neuroanatomical parameters of the spinal cord for a decade . In the As a functional and anatomical unit, neurons of the spinal cord are the final common pathway for the transmission of the neuronal information from a variety of sources to the skeletal muscles . BesidesIt is widely believed that functions are impaired or completely lost depending on the severity of the axonal tract damage, while function recovery relies on the restoration of the corresponding impaired axons . The CSTTo further explore the mechanism of CIG-induced axon growth, certain important molecules  in the myelin-associated inhibition signaling pathway were measured in this study. The axonal growth cone collapse induced by inhibitory molecules associated with myelin is one of the major causes for the inhibition of the axon regeneration . Nogo-A In conclusion, the current study demonstrated that intragastric administration of CIG ameliorated locomotor impairment, reduced the LEp volume, increased the spared tissue (GM and WM) and neuron survival, enhanced biofunctional axons growth and their passage across the lesion site, and downregulated Nogo-A, p75NTR, and ROCKII expression in the myelin-associated inhibition signaling pathway in the injured spinal cords in rats. The results suggest that CIG may be beneficial to SCI treatment.


The laparoscopic approach has become increasingly incorporated into the development of new surgical procedures. An ever-increasing number of surgeons desire methods that minimize surgical trauma and provide improved cosmetic outcomes. Since 2014, we have performed two-port laparoscopic surgery using a transumbilical multichannel glove port and a 12-mm port. The aim of this study was to compare the short-term surgical results of two-port laparoscopic anterior resection (TPLAR) with those of conventional laparoscopic anterior resection (CLAR) for rectal cancer.Between January 2014 and May 2014, a total of 27 patients underwent TPLAR and 30 patients underwent CLAR for the treatment of rectal cancer. The short-term surgical results of these two groups of patients were analyzed retrospectively.p < 0.001). The respective median (range) postoperative pain scores were lower in the TPLAR than in the CLAR patients at 24 h (4 (1–6) h vs 5 (2–8) h; p = 0.045), 48 h (3 (1–4) h vs 4 (range 1–8) h; P = 0.004) and 72 h (1 (0–3) h vs 2 (1–5) h; p = 0.010). The median overall score on the satisfaction-with-abdominal-incision questionnaire of the TPLAR patients was significantly higher (better) than the score of the CLAR patients.The differences in operative time, blood loss, conversion rate, complication rate, distal resection margin, number of harvested lymph nodes, duration until ambulation, duration until first flatus, length of postoperative hospital stay, and overall hospital costs between the two groups were not significant. The median (range) length of the abdominal incisions of the TPLAR patients was shorter than the length of the CLAR patients (5.1 (4.5–16.3) cm vs 8.2 (7.0–10.0) cm, respectively; TPLAR for rectal cancer is safe and feasible, with short-term perioperative and oncological outcomes similar to those of CLAR. TPLAR provides less postoperative pain and better cosmetic outcomes. Laparoscopic surgery is widely used for surgical procedures. Procedures using the laparoscopic approach have even become the standard for many abdominal operations, because of the benefits of reduced blood loss, faster postoperative recovery, shorter hospital stay, reduced postoperative pain, and improved cosmetic outcomes compared with open surgery –5.Colorectal cancer is a common malignant tumor, and surgical resection remains the primary method of treatment. Ongoing improvements in laparoscopic techniques have led to the increasing use of laparoscopic surgery for the treatment of colorectal cancer. Many studies have confirmed that laparoscopic surgery provides oncological outcomes comparable to those of open surgery –8.Conventional laparoscopic colorectal surgery is usually performed with five ports. In the pursuit of minimal invasiveness, colorectal surgeons modified the five-port procedure by developing reduced-port laparoscopic surgery (RPLS) and single-incision laparoscopic surgery (SILS). However, SILS can be challenging to perform, requiring specially designed instruments and having a long learning curve. RPLS is a compromise and appears to be suitable for a variety of applications. In addition, SILS and RPLS usually employ a transumbilical incision for the placement of a specific multichannel device for the insertion of instruments and the laparoscope. Most commercially available multichannel devices are expensive. Here, we introduce a simple, inexpensive, and easily available self-made device that consists of a wound retractor and a surgical glove.The aim of this study was to compare the short-term surgical results of two-port laparoscopic anterior resection (TPLAR) performed via a transumbilical multichannel glove port and a 12-mm port vs conventional laparoscopic anterior resection (CLAR) for rectal cancer.4 cancer; (2) American Society of Anesthesiologists (ASA) classes IV and V; (3) anticipated need of intensive care; (4) history of previous major abdominal surgery; (5) computed tomography (CT) or MRI evidence of tumor infiltration of adjacent organs. A total of 57 patients fulfilled the criteria. Of them, 27 patients underwent TPLAR via a 12-mm port and a transumbilical multichannel port constructed from a wound retractor and a surgical glove. They were matched by age, gender, body mass index (BMI), ASA class, history of abdominal surgery, tumor location, and preoperative TNM stage with 30 patients who underwent CLAR.This was a retrospective study of patients with rectal cancer who were treated at the Department of Colorectal Surgery of the Shengjing Hospital of China Medical University. Between January 2014 and May 2014, a total of 73 patients underwent surgery for rectal cancer. Patients who had nonmetastatic rectal cancer and required anterior resection were included. The exclusion criteria included the following: (1) preoperative magnetic resonance imaging (MRI) showing a tumor of >4 cm in diameter, evidence of local invasion, or TAll patients underwent a preoperative diagnostic workup that included digital rectal examination, complete colonoscopy, serum carcinoembryonic antigen level, and tumor staging  . During All the procedures were performed by the same experienced surgical team. Under general anesthesia, all patients were placed in the modified lithotomy position. For TPLAR, a 3–5-cm transumbilical incision was made, and a self-made transumbilical multichannel device that was constructed from a wound retractor  and a surgical glove Fig.  was inseFor CLAR, the procedures were performed via five ports, and most procedures were similar to those used for TPLAR; however, the specimen was extracted through an enlarged umbilical incision.All patients were treated by the same standardized protocol after surgery. Each patient was allowed to drink 50 mL of water per day from the first day after surgery until passage of flatus and then was allowed unlimited oral intake of fluids. The patient was allowed a solid diet after the passage of feces. Each patient chewed gum three times daily from the morning of postoperative day 1 until the first passage of stool. Patients were given postoperative intravenous antibiotics for 3–5 days. We encouraged early ambulation and moderate activity. Postoperative analgesia consisted of intravenous parecoxib sodium 40 mg every 12 h for 3 days.Satisfaction-with-abdominal-incision questionnaire (SAIQ) is a five-item questionnaire that is used to assess the patient’s satisfaction with the abdominal incisions Table . These iThe following types of data were analyzed: patient and tumor characteristics, procedural details and perioperative data, postoperative pain score, length of postoperative hospital stay, and SAIQ scores. Postoperative pain was measured using a visual analogue scale (VAS) at 12, 24, 48, 72, 96, and 120 h after surgery. Postoperative complications included bleeding, anastomotic leakage, wound infection, intraabdominal abscess, ileus, and urinary retention. All patients were asked to complete the SAIQ, which was administered by a nurse, 1 day before or the day of discharge from the hospital. The overall direct medical costs were calculated for each patient group. The patients were followed by the outpatient department at 1-month intervals for the first 3 months after discharge from the hospital and at 3-month intervals thereafter for longer than 1 year. We contacted some patients by phone to confirm the validity of the follow-up data.U test. Discrete variables were compared by the chi-square test or Fisher exact test. All statistical calculations were performed using SPSS software, version 19.0 . A p value ≤0.05 was considered statistically significant.Results are expressed as median (range) or rates (%). Continuous variables were analyzed using the Mann-Whitney p > 0.05 for all comparisons).Fifty-seven patients with primary rectal cancer, who underwent anterior resection between January 2014 and May 2014, were enrolled in our study (27 TPLAR and 30 CLAR patients). Patient and tumor characteristics are presented in Table p = 0.099). The difference in median volumes of blood lost was not statistically significant (p = 0.084). One TPLAR patient was converted to open surgery because of abdominal adhesions, and no patients were converted to conventional laparoscopic surgery. No CLAR patients were converted to open surgery. The difference in rates of open surgical conversion was not statistically significant (p = 0.474). No intraoperative complications occurred during any of the procedures. All of the abdominal incisions, including transumbilical incision, trocar sites, enlarged umbilical incision, and open surgical incision were measured. The median length of the abdominal incision was statistically significantly shorter in the TPLAR group than in the CLAR group (5.1 (4.5–16.3) cm vs 8.2 (7.0–10.0) cm; p < 0.001). The differences in the median width of the distal resection margins (p = 0.993), median number of harvested lymph nodes (p = 0.086), and the percentages of patients with each TNM stage (p = 0.488) were not statistically significant. The overall postoperative complication rate was 3.7 % in the TPLAR group and 10 % in the CLAR group (p = 0.613). One TPLAR patient developed postoperative urinary retention, and three CLAR patients developed postoperative complications (ileus (n = 1), urinary tract infection (n = 1), and urinary retention (n = 1)). None of the patients with complications underwent additional surgery for their postoperative complication. The differences in median duration until ambulation (p = 0.261), duration until first flatus (p = 0.243), length of postoperative hospital stay (p = 0.060), and overall hospital costs (p = 0.676) between the two groups of patients were not statistically significant. None of the patients in either group was rehospitalized during the first 30 postoperative days. In addition, none of the patients developed hernia at the trocar sites, metastasis to the incisions, or local recurrences. None of the patients died during the follow-up period.Procedural details and perioperative outcomes are provided in Table p = 0.045), 48 h (3 (1–4) vs 4 (1-8) h; p = 0.004), and 72 h (1 (0–3) h vs 2 (1-5); p = 0.010). The differences in postoperative pain scores were not statistically significant at 12, 96, and 120 h.The postoperative pain scores of both TPLAR and CLAR patients dropped rapidly Fig. . Table 4p = 0.001). The differences between patient groups in the median scores for length (p = 0.017), esthetics (p = 0.030), and pain (p = 0.003) were statistically significant. However, the differences in the median scores for position and healing of incisions were not significant (Fig. The outcomes of the SAIQ are detailed in Table s p = 0.00, and paLaparoscopic surgery for rectal cancer has been performed worldwide. The COLOR II trial reported that laparoscopic surgery provided rates of locoregional recurrence and disease-free and overall survival similar to those of open surgery . The proSeveral authors have reported that RPLS for colorectal cancer is feasible and safe –20. In gIn some of the previous studies, most of the surgeons made a transumbilical incision for the placement of a commercially available multichannel system, and then an additional 5- or 12-mm trocar was placed in an incision in the right lower quadrant –18. HoweA total of 27 patients underwent TPLAR for rectal cancer in our hospital at the time of this report. We evaluated and compared the short-term surgical results of the patients undergoing TPLAR with the results of 30 patients who underwent CLAR for rectal cancer. The operative outcomes, including operative time, blood loss, conversion rate, complication rate, and postoperative recovery were similar between the two patient groups. The oncological adequacy of TPLAR was a concern. However, in our study, the differences in sizes of distal resection margin and number of harvested lymph nodes were not significant. In addition, none of the patients in either group developed local recurrence or died during the follow-up period. Our results demonstrate that TPLAR for rectal cancer can provide satisfactory short-tem oncological outcomes, which were comparable to those reported by Bae et al. .Our study found that the mean length of abdominal incisions in the TPLAR group was significantly shorter than the mean length in the CLAR group. Lim et al. reported that differences between the lengths of umbilical incisions resulting from umbilical-incision laparoscopic colorectal cancer surgery using a single additional port and from conventional laparoscopic surgery were not significant . The selThree case-matched studies , 27, 28 To the best of our knowledge, there have not been any reports on the use of a gold-standard scoring system for evaluating the degree of patient satisfaction with abdominal incisions until now. Arrigoni et al. reported on a quality-of-life questionnaire using a five-point Likert scale that included the determination of patient satisfaction with wound cosmesis . HoweverThe placement of a tube into the pelvic cavity after rectal surgery in order to drain potential postoperative blood loss and anastomotic leakage is controversial. Notably, pelvic drainage is not recommended by the Society of Enhanced Recovery After Surgery . HoweverThis study has limitations. First, the numbers of patients in the two groups were small. Second, our study had a short follow-up time. We therefore could not evaluate the long-term oncological results and determine the incidence of late complications, such as trocar-site hernia and metastasis to the incisional sites for TPLAR vs CLAR. And third, the colorectal surgeons in this study had performed more than 500 CLARs for rectal cancer before they began using two-port procedures. The difference in proficiency between the two techniques might have led to a small bias.In conclusion, two-port laparoscopic technology can overcome some of the shortcomings of conventional laparoscopic surgery and SILS, while also providing some of their benefits. TPLAR for rectal cancer, as performed by expert laparoscopic surgeons, is safe and feasible. The short-term perioperative and oncological outcomes were similar to those of CLAR. TPLAR provides less postoperative pain and better cosmetic outcomes. In addition, our self-made multichannel device is simple, cheap, and easily available, and can reduce costs. However, additional well-designed randomized studies with larger numbers of patients are needed for evaluating the long-term oncological results of our procedure.


Recent reports in human demonstrate a role of theta–gamma coupling in memory for spatial episodes and a lack of coupling in people experiencing temporal lobe epilepsy, but the mechanisms are unknown. Using multisite silicon probe recordings of epileptic rats engaged in episodic-like object recognition tasks, we sought to evaluate the role of theta–gamma coupling in the absence of epileptiform activities. Our data reveal a specific association between theta–gamma (30–60 Hz) coupling at the proximal stratum radiatum of CA1 and spatial memory deficits. We targeted the microcircuit mechanisms with a novel approach to identify putative interneuronal types in tetrode recordings  and validated classification criteria in the epileptic context with neurochemical identification of intracellularly recorded cells. In epileptic rats, putative parvalbumin basket cells fired poorly modulated at the falling theta phase, consistent with weaker inputs from Schaffer collaterals and attenuated gamma oscillations, as evaluated by theta-phase decomposition of current–source density signals. We propose that theta–gamma interneuronal rhythmopathies of the temporal lobe are intimately related to episodic memory dysfunction in this condition. Cognitive deficits of temporal lobe epilepsy represent a major comorbidity of this disease, but how epileptogenic microcircuits serve cognitive demands during seizure free periods remain poorly understood. Here, we investigate this issue using an experimental rat model of temporal lobe epilepsy. Our data link hippocampal theta phase-specific uncoupling in the slow gamma band with disruption of episodic-like memory function and microcircuit deficits resulting from poor oscillatory firing of parvalbumin basket cells.Interaction between brain oscillators has been revealed as a critical mechanism to orchestrate synchronization between neuronal circuits . One exaIn the hippocampus, different gamma generators couple preferentially to distinct theta phases. Mid-gamma generator (60–90 Hz) operates at the peak of the CA1 hippocampal theta oscillation recorded at the stratum pyramidale SP;  in coordTemporal lobe epilepsy (TLE), a disease affecting the hippocampus and parahippocampal regions, is associated with a range of cognitive comorbidities, and episodic memory deficits in particular . AlthougHere, we demonstrate that disruption of theta–gamma coordination in the dorsal hippocampus of TLE rats is linked to specific aspects of episodic-like memory deficits in these animals in periods free of seizures or interictal activities. We elucidated the mechanisms of this phenomenon with a combination of multisite and single-cell recordings, and found that dysfunction of parvalbumin (PV) basket cells, a resilient interneuronal population in TLE, may be central to this form of hippocampal rhythmopathy.All experimental protocols and procedures were performed according to the Spanish legislation (R.D. 1201/2005 and L.32/2007), and the European Communities Council Directives of 1986 (86/609/EEC) and 2003 (2003/65/CE) for animal research, and were approved by the Ethics Committee of the Instituto Cajal and the Spanish National Research Council.Adult male Wistar rats (180–200 g) were treated with multiple intraperitoneal injections of kainate (5 mg/kg) at hourly intervals until they reached status epilepticus. Diazepam (4 mg/kg) was injected 1 h later to stop convulsions. Animals received intraperitoneal injections of 2.5 ml of 5% dextrose in saline, and their diet was supplemented with fruit and powder milk during the following 2–3 d. After 4–5 d post-status, rats behaved normally and were housed individually. Some rats were injected with saline instead of kainate and received treatments similar to those in epileptic animals. Untreated normal rats completed the control group, since no differences between them and saline-injected animals were detected in electrophysiological indices. All quantitative experiments started 8 weeks after status when rats already exhibited recurrent seizures being in the chronic phase. Seizures were either observed during animal handling or recorded electrophysiologically during behavioral tasks. In epileptic rats, periods of normal-like electrophysiological activity were intermixed with periods dominated by epileptiform events, defined as convulsive or subclinical seizures and interictal discharges . Only daA total of 18 normal rats  and 17 epileptic rats were used in the study.2) were implanted either fixed or mounted on an adjustable microdrive (either custom-made or the nDrive from NeuroNexus). For tetrode recordings, microdrives  with eight independent screws loaded with tetrodes  were used. All electrodes were implanted perpendicular to the skull surface. Two screws served as a reference and ground at the occipital region. After surgery, rats received short-term treatment of enrofloxacin 10 mg/kg, methylprednisolone 10 mg/kg, and buprenorphine 0.05 mg/kg.To implant either multisite silicon probes or tetrodes, rats were anesthetized with isoflurane (1.5–2%) in oxygen (30%) and were continuously monitored with an oximeter . Epileptic and saline-treated rats were implanted 6–7 weeks after treatment to allow for at least 1 week of recovery and stabilization before quantitative experiments started. Implantation coordinates ranged from 3.9 to 6 mm posterior to bregma, and between 2 and 5 mm from midline. Sixteen-channel silicon probes  was estimated from an infrared LED coupled to the headstage and imaged from a ceiling camera .After recovering, electrophysiological recordings were obtained using a multichannel amplifier . Local field potential (LFP) signals from multisite silicon probes were acquired with a 16-channel headstage, amplified by 400 and stored at 4800 Hz/24 bit precision in a 1–2400 Hz frequency band after analog filter. Signals from tetrodes were obtained with 32-channel headstages, amplified (400× to 1000×), bandpass filtered , and stored at 24 kHz/24 bit precision. In all cases, the position of the rat in the arena  over consecutive days for a given animal before starting experiments. In animals carrying microdrives, the position of the silicon probe was adjusted in depth to target similar layers over different days. For tetrodes, screening was performed to check for the ability to record units and to infer the electrode position by lowering each tetrode independently toward the hippocampus. Every day, single tetrodes were advanced individually until unit-firing and recognizable hippocampal activities  were identified. Recordings were obtained daily from Monday to Friday between 2:00 and 7:00 P.M. over 1–3 weeks while the animals performed different cognitive tasks.f spectral behavior . For tetrode recordings, three control rats (untreated) and three epileptic rats were used.We implanted silicon probes in 10 control rats  and 10 epileptic rats. After completing experiments, some of these rats were recorded in the dead condition (after overdose of anesthesia) with the silicon probe in place before fixation, to evaluate aspects of the 1/We used the “what-where-when” paradigm of object recognition tasks to test for episodic-like memory in TLE rats . The tesDuring the task, animals encountered four copies of an object during a first sample phase . Objects were chosen after confirming no specific preferences, and that they were big enough to avoid climbing or dropping . A second sample phase (3 min duration) followed 50 min after the first, in which rats found four copies of another object , presumably reflecting a temporal component of episodic-like memory, and for the recent familiar displaced object (B2), reflecting spatial memory for events . The what test). We also defined an index of the spatial memory component (the “where” index) as the proportion of time spent exploring the displaced object B2 against the other stationary object seen in sample phase 2 divided by the total time spent with them  independently . In thisFor these experiments, six control rats  and six epileptic rats implanted with silicon probes were tested in the what-where-when task. We also tested four control rats  and four epileptic rats in the one sample where task. Two epileptic rats and one control rat were tested in both tasks. In addition, for rats implanted with tetrodes, we ran the what-where-when task in one control rat (untreated) and one epileptic rat, and the where task in two control rats (untreated) and two epileptic rats. Remarkably, all these epileptic rats were free of epileptiform activities within 2 h of the tasks. In four epileptic rats, seizures and/or interictal activities were recorded during the what-where-when task, and they were analyzed separately. In three of these animals, episodic-like memory was retested on a different day in the absence of any sign of epileptiform activities. All memory tasks were performed at equivalent times of day.We used sessions of free exploration of different arenas without objects  to screen hippocampal layers and units. These sessions were intermixed with the object recognition tasks, either as habituation sessions for the task itself or as independent screening sessions. After completing object recognition tasks, we ran standard pellet-chasing sessions to improve unit sampling from tetrodes and to eventually establish their place cell behavior. To this purpose, rats implanted with tetrodes were deprived of food for up to 85–90% of their original weight. Initially, animals were allowed to explore freely the open field over 3–5 min. During each session (10–15 min), small pellets of food dropped in every 20 s to random locations within the open field, keeping the animal in continuous locomotion and allowing a full sampling of the arena. Electroencephalographic recordings were obtained continuously during free exploratory and pellet-chasing sessions.All rats implanted with tetrodes were tested with these tasks . Some rats implanted with silicon probes were tested with pellet chasing, but data are not discussed .www.octave.org). One session (300 s) free of artifacts (mostly due to interactions between headstage and objects during the exploration of the rat) and with >14 functional channels spanning around SR and SLM were selected from the analysis of multisite LFPs from each animal. Current source density (CSD) signals were calculated using the second spatial derivative of LFPs at 100 µm resolution. Smoothing and defective site interpolation were used for visualization purposes only. CSD signals from the SP layer could not be consistently evaluated since they were typically sampled by the first recording channel or by none. LFP signals from tetrodes were analyzed similarly.Signals were analyzed using routines written in MATLAB (MathWorks version 10b) and/or Octave  for each rat, suggesting that they capture basal spectral features of hippocampal microcircuits  with continuous theta were identified from different recording sessions (300 s each) using spectral criteria, as previously described . Controlcircuits . For viscircuits C, power The positive peak of theta waves at SP was used as a reference to study theta–gamma modulation using phase-decomposition approaches . To estiTo evaluate theta-phase coupling of gamma activity, the following phases were defined with respect to the theta peak at SPk, as follows: −2π, −3/2π, −π, −π/2, 0, π/2, π, 3/2π, and 2π. Phase segments were defined based on the average cycle duration. The FFT was calculated for each phase segment, and detrended in each session considering all of the available segments. The 1/f behaviour of LFP spectrum led us to make use of these detrended Fourier coefficients instead of other phase-amplitude measurements . Data frA modulation index was defined for each gamma band by estimating the difference between the maximal and the minimal gamma power modulated across the theta cycle using data from −π to π. To test the modulation index statistically, we chose a conservative surrogate design that destroyed phase-specific gamma coupling while preserving unspecific nonstationarities of the original data. In preliminary tests, we noted that phase segment shuffling resulted in equalized gamma activity across the theta cycle. To avoid this effect, we cut the 13 phase segments at random points and exchanged the resulting two phase vectors along 5000 realizations, thus preserving the original nonstationarities . In multSingle units were isolated from tetrode recordings using cluster-cutting techniques . The initial discrimination of clusters and manual refinement was performed using principal components, spike amplitude, and other parameters. To verify cluster quality isolation, the probability of cluster overlapping was calculated as described previously . Spike tWe evaluated theta phase-locking behavior of unit firing by using phase histograms of spike timing around theta peaks recorded at SP. Phase histograms were represented as firing probability from −2π to 2π by dividing each phase bin (8.7º bin size) by the total number of spikes. Phase locking was quantified using the mean vector length of phase distribution, which is a measure of phase consistency between spikes and LFP from 0 to 1. A minimum of 1000 spikes was required to estimate this measure accurately.To evaluate theta rhythmicity of unit firing, we estimated the power spectrum of the autocorrelogram built at ±0.5 s windows (1 ms bin size). A theta autocorrelation index was defined from the normalized area in the 4–12 Hz band. We also evaluated gamma rhythmicity similarly, by computing the power spectrum of autocorrelograms in the 40–90 Hz band to define a gamma autocorrelation index.We ran simulations of theta phase-modulated firing to evaluate the sensitivity of indices of theta phase locking . Spikes (2000–2500 spikes in a 300 s window) with different degrees of phase locking to a sinusoidal theta wave (8–9 Hz) were used. We evaluated the effect of mean firing rate reduction in the synthetic time series by randomly deleting a fixed proportion of individual spikes every run (100 simulation runs). The large number of spikes (>1500) used ensures a poor effect of spike counts on measures. Phase and autocorrelogram distribution histograms similar to those used for unit data were computed for each simulation run. The mean ± 95% confidence interval value of the phase vector length and the autocorrelation index were reported.t test (p < 0.05). We also exploited the behavior-dependent specialization of identified interneurons to subclassify them according to their firing dynamics during run/stop transitions  basket cells, and oriens lacunosum moleculare (OLM) and bistratified interneurons based on the following criteria: PV basket cells fired in the descending phase of theta and had SPW/ripple ratios that were significantly >1 and run/stop ratios were significantly >1 ; CCK basInterneurons that did not meet these criteria were not subclassified. Classification was performed manually based on the quantitative criteria described above.m potassium acetate and 2% Neurobiotin (Vector Laboratories) for subsequent histological verification. Recorded interneurons  were successfully recovered for histological studies, including immunostaining analysis in five control rats (untreated) and four epileptic rats.To validate some of these classification criteria in the TLE context, we obtained intracellular recordings in urethane-anesthetized normal and epileptic rats (1.2 g/kg), as previously reported . Brieflym, pH 7.4 PBS. Brains were postfixed overnight at room temperature (RT), washed in PBS, and serially cut in 70 µm coronal sections . Sections containing the probe and tetrode tracks were identified with a stereomicroscope . Sections containing Neurobiotin-labeled cells were localized by incubating them in 1:400 Alexa Fluor 488-conjugated streptavidin  with 0.5% Triton X-100 in PBS (PBS-Tx) for 2 h. Sections were analyzed using an inverted epifluorescence microscope . Those containing the recorded soma were selected for immunofluorescence characterization.After completing recordings, rats were transcardially perfused with 4% paraformaldehyde and 15% saturated picric acid in 0.1 For immunohistochemistry, we used a battery of antibodies including monoclonal anti-NeuN , polyclonal anti-netrinG1 (1:10) and anti-netrinG2 , polyclonal rabbit anti-PCP4 , monoclonal mouse anti-PV , and polyclonal rabbit anti-somatostatin-14 . Some sections were also stained with bisbenzimide or a Nissl reaction with toluidine blue. Immunostaining was performed on floating sections using either the biotin–avidin–peroxidase method or fluorescent secondary antibodies.For silicon probe recordings, NeuN and netrins G1 and G2 were used to identify electrode tracks across layers and regions . For tetThe identity of intracellularly recorded cells was confirmed by colocalization of different neurochemical markers and streptavidin. Sections containing the somata of the recorded cell were immunostained against PV and somatostatin overnight followed by incubation for 2 h at RT with appropriate secondary antibodies including goat anti-rabbit Alexa Fluor 633 , and goat anti-mouse Rhodamine Red  in PBS-Tx and 1% FBS. Streptavidin-positive recorded cells were identified using a confocal microscope , as previously described .Statistical analysis was performed either with the SPSS software (IBM) or with MATLAB. No statistical methods were used to predetermine sample sizes, but they were similar to those reported previously .t tests. Correlation between electrophysiological indices or spectral and behavioral indices was evaluated with the Pearson product-moment correlation coefficient, which was tested against 0  at p < 0.05 (two sided). Both the Pearson coefficient and p value are reported to facilitate interpretation.Normality was confirmed for spectral indices (either directly or after logarithmic transformation) using the Kolmogorov–Smirnov test. Homoscedasticity was tested using the Levene’s test. Gamma power values from each band  were analyzed with ANOVAs of various factors . To quantify theta–gamma modulation power measurements estimated from LFP and CSD, signals from each gamma band were compared among groups, layers, and location for a given band, and between bands and groups from different layers and phases. Theta phases were restricted to the four central phases around the peak recorded at SP , to include only one theta cycle in the analysis. Comparisons between groups and proximodistal location were evaluated using Welch’s t tests. Rayleigh circular statistics, equivalent to the t test, was used to characterize phase-locking behavior whenever required. The phase-locking vector length and theta and gamma autocorrelation indices from units were analyzed with ANOVAs for groups and recording locations, after confirming normality.Normality and homoscedasticity (Levene’s test) were not confirmed for firing rate and theta-phase preference data of units sorted from tetrodes. They were analyzed with the Bartlett’s test for variance, and posteriorly compared with Welch’s Results are represented as the mean ± SD for indices meeting the normality test. Results are represented as whisker-box plots or as mean ± SD values with individual points when normality distribution was not confirmed.r > 0.7, p < 0.01 for between-session comparisons; To examine layer-specific theta–gamma coupling, we used 16-channel silicon probes to record LFPs from rats engaged in object recognition tasks that tested for the spatial and temporal aspects of episodic-like memory  = 52, p < 0.0001; epileptic rats: F = 61, p < 0.0001), possibly reflecting that distinct gamma generators contribute unevenly to scale-free dynamics across different layers . Overall, the absolute power poorly accounted for group differences across gamma bands .Examination of the distribution of LFP gamma activity across layers showed a typical laminar profile both in control and epileptic rats C. BroadbF = 25.1; p = 0.0004; in three rats, the SP was not sampled) and a not significant trend in SR  = 4.1; p = 0.063; Post hoc Bonferroni-corrected Welch’s t tests suggested increased fast gamma activity in epileptic rats both at proximal and distal locations  = 5.9, p = 0.0293; proximodistal effect: F = 15.5, p = 0.0015; interaction, p = 0.0067), but these were associated with a decreased gamma power at proximal locations instead  = 4.8, p = 0.0451 for groups; no proximodistal effects but significant interaction at p = 0.0013), suggesting that increased fast gamma activity at SP may be contaminated by neuronal firing  = 30.5, p < 0.0001; F = 8.4, p = 0.0053; F = 4.8, p = 0.0373; F = 17.3, p = 0.0027; p < 0.00001) and epileptic rats (p < 0.001). Importantly, we detected group differences of modulation indices at proximal locations for the slow gamma band  = 3.7, p = 0.0445; two-way ANOVA for groups and location) but not for mid-gamma band  = 5.7; p = 0.012; r = 0.83, p = 0.011) and in both groups  oscillations at the proximal SR in TLE rats. Theta-phase preference of CA1 gamma band has been associated with specific mechanisms of CA3 and entorhinal cortical inputs during the encoding and retrieval of information . We thusCognitive associates of theta–gamma disruption were investigated by examining the correlation between spectral and cognitive indices derived from the episodic-like memory task (what-where-when) in six control rats and six epileptic rats . During these tasks, rats discriminate between objects carrying different degrees of novelty A, requirn = 6) explored similarly to control rats , animals tended to be less exploratory compared with their epileptic mates  and B2 (p = 0.0293). The other two objects (p = 0.0013) and when indices  of the spatial and temporal components of recognition memory  and for both groups together . We sorted 87 PCs  and 23 interneurons  from control animals. A total of 66 PCs  and 20 interneurons  were sorted from epileptic rats. Some units were left unclassified  and were not included in the analysis , similar to recent reports (p = 0.6189). A Bartlett’s test for variance across groups and locations suggested a strong difference in the variability of firing rate values of PCs (×2(3) = 17.9, p = 0.0005). Despite this variability, we found a lower mean firing rate in proximal PCs of the epileptic group compared with those in the control group . No group (p = 0.1340) or proximodistal effects (p = 0.2898) were confirmed for putative interneurons, which exhibited large variability within groups . The phase vector length failed to capture differences between groups and exhibited large variability  = 6.8, p = 0.0163; F = 5.9, p = 0.0168), but not group effects, dominated statistical differences (no interaction).We noted different phase-locking behavior, especially for interneurons in epileptic rats . We chose to evaluate the effect of reducing the mean firing rate to simulate trends detected in control and epileptic rats D. The phThus, our data suggest that theta-modulated firing of proximal CA1 interneurons is reduced in the epileptic hippocampus, pointing to specific mechanisms. However, different sensitivities of modulation indices and individual variability between interneuronal types precluded strong group differences. Given the role of proximal PV basket cells in slow gamma oscillations at the falling theta phase , we askePV-positive interneurons represent a major population involved in the generation of gamma oscillations . ImportaTo evaluate this point further, we exploited cell type-specific firing features and subclassified putative interneurons recorded with tetrodes in four different classes . This approach was adopted previously , but, inn = 4) and OLM interneurons (n = 2) exhibited separate behavior during run/stop transitions (n = 2) and bistratified interneurons (n = 2), while the remaining putative interneurons could not be unequivocally subclassified , suggesting their weak feedforward activation by Schaffer collaterals. Thus, we validated our classification criteria for PV basket cells in the TLE context with intracellular recordings from urethane-anesthetized rats . PV basket cells were neurochemically identified in one of five (control) and one of four (epileptic) interneurons. They were immunoreactive to PV but not to somatostatin and had several synaptic boutons around pyramidal cell bodies  oscillations are attenuated in the proximal CA1 region of epileptic rats. Strikingly, poorly modulated slow gamma oscillations at SR explain part of the variability of spatial memory abilities in an episodic-like memory task. The identification of putative GABAergic interneurons, and PV basket cells in particular, suggest that the disruption of their firing at the falling theta phase may be at the basis of this rhythmopathy. Thus, our data suggest that in periods free of interictal and ictal activities, cognitive comorbidities of TLE may be intimately related to specific microcircuit dysfunction in this neurological condition.Gamma oscillations are supposed to emerge locally from either interneuron–interneuron (ING models) or pyramidal cell–interneuron interactions  at the falling theta phase was reduced and poorly modulated in TLE animals E,F. RecePoor rhythmicity of PV basket cells during theta oscillations may result from many factors. Reduced synaptic recruitment of specific classes of interneurons is reported in TLE , suggestWe found that variability in theta phase modulation of the slow gamma (30–60 Hz) correlates with spatial memory deficits of epileptic rats in the episodic-like memory task 4E. This Although our current understanding of the role of hippocampal oscillations in cognition and the mechanisms underlying is yet limited, data suggest there are strong links. We have identified a microcircuit rhythmopathy of theta–gamma coupling at the slow band in the proximal SR of TLE rats. Poor rhythmicity of PV basket cells appears to be at the basis of this impairment. In models of Alzheimer’s disease, a disease linked to early-onset temporal lobe epilepsy and reduced gamma activity alteration, the excitability of PV interneurons has been associated with similar cognitive deficits . We prop


Plasmodium falciparum AMA-1, MSP-119 and CSP were measured with the aim to describe transmission patterns in meso-endemic settings in Lake Victoria. Two cross-sectional surveys were conducted in Lake Victoria in January and August 2012. The study area comprised of three settings: mainland (Ungoye), large island (Mfangano) and small islands . Individuals provided a finger-blood sample to assess malaria infection by microscopy and PCR. Antibody response to P. falciparum was determined in 4,112 individuals by ELISA using eluted dried blood from filter paper. The overall seroprevalence was 64.0% for AMA-1, 39.5% for MSP-119, and 12.9% for CSP. Between settings, seroprevalences for merozoite antigens were similar between Ungoye and Mfangano, but higher when compared to the small islands. For AMA-1, the seroconversion rates (SCRs) ranged from 0.121 (Ngodhe) to 0.202 (Ungoye), and were strongly correlated to parasite prevalence. We observed heterogeneity in serological indices across study sites in Lake Victoria. These data suggest that AMA-1 and MSP-119 sero-epidemiological analysis may provide further evidence in assessing variation in malaria exposure and evaluating malaria control efforts in high endemic area.As markers of exposure anti-malaria antibody responses can help characterise heterogeneity in malaria transmission. In the present study antibody responses to  Nevertheless, with scaling up of malaria prevention, diagnosis and treatment, the prevalence of Plasmodium falciparum infection in many parts of sub-Saharan Africa declined by 50%, and the incidence of clinical disease fell by 40% between 2000 and 20152.In sub-Saharan Africa, malaria remains one of the leading causes of morbidity and mortality, with 191 million cases and over 390 thousand deaths reported in 2016P. falciparum parasite rate for the population aged 2–10 years (PfPR2–10) is below 1%3. Despite significant increases in coverage of long-lasting insecticide-treated nets (LLINs) through free-mass distribution campaigns4 and the change in first-line treatment to artemisinin-based combination therapy (ACT)5, 10.6% (4.3 million) of the population still live in areas with PfPR2–10 ≥ 40%, mainly counties adjacent to Lake Victoria in western Kenya6. In addition, increased paediatric malaria hospital admissions in 20097 and high prevalence of malaria infection among school children in 20148 highlight the challenge of effective malaria control in this highly endemic region of Kenya.In Kenya, 65% (26 million) of the population live in areas where 10. As a proxy measure of malaria transmission, serological responses to P. falciparum antigens have shown a robust and consistent correlation with estimates of entomological inoculation rate (EIR)10, and thus have increasingly been incorporated in cross-sectional and longitudinal studies to monitor changes in transmission15 and identify hotspots in transmission17.Understanding the intensity of malaria transmission in a community is fundamental to the design and evaluation of malaria control and elimination programmes. The most widely used metric of malaria transmission intensity is parasite prevalence, measured through cross-sectional surveys. In recent years, antibody responses to one or more malaria parasite-specific antigens have been explored as alternative means to estimate malaria transmission intensity20, no such study has been carried out in the adjacent Lake Victoria basin where prevalence is moderate to high with significant local heterogeneity21. In the present study, antibody responses to P. falciparum blood-stages antigens apical membrane antigen 1 (AMA-1), merozoite surface antigen-119 (MSP-119) and circumsporozoite antigen (CSP) were measured to assess malaria exposure and transmission on islands in Lake Victoria. Results from this study provide baseline data to evaluate the planned malaria elimination programme in the study area.Whilst several sero-epidemiological studies have been conducted in the low-transmission western highlands of KenyaA total of 5044 participants were enrolled from five different settings  in January and August 2012. Population coverage varied among settings: 10.5% in Mfangano, 35.7% in Ungoye and 48–90.6% in the small islands. Gender and age distributions were similar across the five settings. The majority of participants were children and adolescents ≤15 years old  and came from the islands . At enrolment, 5.9% (95% CI: 5.2–6.5) of the population were febrile (axillary temperature >37.5 °C), and 20.8% (95% CI: 19.7–22.0) were anaemic (haemoglobin [Hb] level < 11 g/dL). Of all children 12 years and below (n = 3045), 1261  were found to have an enlarged spleen. The prevalence of febrile illness, anaemia, and enlarged spleen varied significantly by study sites (P < 0.001). Further details on the study population are shown in Table P. falciparum infection by microscopy and PCR in the study sites is shown in Fig. P. falciparum parasite prevalence ranged between 4.1 and 32.1% by microscopy, and between 11.2 and 56.2% by PCR. Parasite prevalence was significantly higher in Ungoye than other sites, regardless of detection method . Parasite prevalence by PCR generally peaked in the 11–15 years group and declined thereafter in all study sites  for Ungoye, Mfangano, Takawiri, Kibuogi, and Ngodhe, respectively  and PCR   between Ungoye (43.4%) and Mfangano (45.4%), and significantly lower on the three small islands . Seroprevalence between genders showed a higher prevalence in females in Ungoye (P = 0.023) and Mfangano (P = 0.024), but the differences were comparable in the small islands. Antibody level and seroprevalence increased significantly with age (P < 0.001) in all study areas    . The different serological outcomes for CSP all produced similar trends; similar between the study areas, albeit the differences in antibody responses were statistically lower than AMA-1 and MSP-1ns) Fig. , and thens) Fig. . SCRs we86) Fig. .P. falciparum infected individuals. Antibody responses did not differ significantly between the febrile and the non-febrile groups in all study sites, but the proportions of AMA-1 seropositive individuals were significantly higher (P < 0.05) in the non-febrile groups in Ungoye and Mfangano  among children with enlarged spleen than those with non-palpable spleen in Ungoye and Mfangano, although the differences were not consistently observed on the small islands  infections , Kibuogi (P = 0.008), and Ngodhe (P = 0.037) (Table 19 (P < 0.001) and CSP (P = 0.002) on Ngodhe. Age was significantly (P < 0.001) associated with AMA-1 seropositivity in all five study sites, MSP-119 seropositivity in Ungoye (P < 0.001), and CSP seropositivity on Ngodhe (P = 0.024). Significant association between anaemia and MSP-119 seropositivity was observed in Ungoye (P = 0.012) and Mfangano (P = 0.03) only  associated with seropositivity to AMA-1, MSP-1e P = 0.07  were similar between Ungoye and Mfangano, but significantly higher when compared to the three small islands , SCR based on serology can provide a more robust picture of the malaria transmission dynamics in an endemic area. In this setting, in the area with lack evidence of infections by microscopy, serological and molecular tools enabled a more complete understanding of the ongoing transmission and allowed for an examination of risk factors. Furthermore, as a metric of malaria transmission intensity, EIR is notoriously difficult to measure and the heterogeneous distribution of mosquito bites among individuals within a population makes it even more difficult to standardize32. Previously developed log-log calibration curves that relate SCRs to EIR values can provide a straightforward low-cost measure of malaria transmission10. These serology-based EIR estimates, especially those based on MSP-119 SCR, have shown a robust correlation with measured EIR10, and thus allow for clear differentiation of low-, moderate-, and high-transmission areas.Despite differences in immunogenicity, both 36, making it a suitable marker for monitoring historical changes in transmission intensity14. AMA-1 and MSP-119 are erythrocytic-stage antigens; they are produced continuously and in relative abundance because the erythrocytic-stage parasites go through several cycles of multiplication. In contrast, CSP is a pre-erythrocytic-stage antigen and is available to the immune system when a small number of sporozoites shed this antigen into circulation37. In consequence, the availability of CSP to immune system is of much shorter duration, which may explain its inability to detect subtle differences in exposure and transmission among settings in this study.Antibody responses to CSP did not follow the same pattern as those for merozoite antigens; neither seroprevalence nor SCR showed significant difference among settings. CSP has been previously reported to give reliable estimates of malaria endemicity in hyper-endemic areas and reflect the seasonal dynamics of transmissionP. falciparum antibody prevalence increased with age. When analysed as a whole population, only antibody responses to AMA-1 were negatively associated with anaemia in Mfangano, Takawiri and Ngodhe. In contrast, when analysis was restricted in children ≤12 years, antibody responses to MSP-119 were significantly associated with anaemia in Ungoye and Mfangano, whilst all the antigens tested were independently associated with enlarged spleen in all areas. Associations between measured antibody responses to P. falciparum antigens and the risk of malaria have shown to be inconsistent in multiple studies38. Potential reasons for these inconsistencies include differences in the intensity and stability of transmission, allelic variation of specific antigens and IgG subclass switching39, which may explain some of the differences in findings in our study areas. Nevertheless, antibodies to AMA-1 have been associated with protection from clinical malaria in some studies41, but not others43. Some studies have also documented independent association between antibodies to MSP-119 and protection from clinical malaria44, whilst others have not45. Similar to the responses to blood-stage antigens, antibodies to CSP was associated with a higher odds of having an enlarged spleen, and age-adjusted antibody levels showed apparent high responses in children with enlarged spleen in each study area  were disproportionally represented . The population in the study area consists mainly of people of the Luo ethnic group with Dholuo is primarily spoken, as well as the national language of Kiswahili. Population estimates were 18,600 for Mfangano, 1000 for Takawiri, 700 each for Kibuogi and Ngodhe , and 3,471 for Ungoye (2009 Kenya Population and Housing Census). The area is predominantly rural with most residents depend on fishing and traditional small-scale farming as the main occupations21. Most houses are typically made of mud walls with thatched or corrugated iron roofs.The present study was conducted on four islands in Lake Victoria  and one mainland lakeshore area (Ungoye) in Homa Bay County, western Kenya Fig. . Homa Ba55. Malaria in the study area is meso-endemic but geographically heterogeneous, with prevalence estimates ranging from 14.6% on islands to 44.2% in the coastal mainland21. The major vector responsible for malaria transmission in Lake Victoria is Anopheles gambiae s.l.56, and estimated annual EIR from serological markers were reported as high as 50 infectious bites per person per year14. P. falciparum is the predominant malaria parasite, while Plasmodium malariae and Plasmodium ovale are less common, and no Plasmodium vivax infections are observed21.The climate in Lake Victoria is tropical, with temperatures ranging from a mean annual minimum of 17.7 °C to a mean annual maximum of 34.8 °C, and humidity is relatively high. This region generally experiences a bimodal pattern of rainfall, with the longer rainy season starting in March and ending in July and the shorter rainy season from November to December. In Lake Victoria basin, peak malaria transmission occurs 1–2 months after the rainy season and the mean monthly anopheline vector abundance has been reported to increase by 6- to 8-fold in the rainy season compared to the dry seasonIn this study, seven catchment areas on Mfangano (i.e. large island) were selected to represent the different local environments found on the island. Gulwe and Kagungu are situated in the central highland. On the other hand, Ramba, Wakinga, Mrongo, Ugina and Wakula are situated along the coastal lowlands of the island. Across the island, there are six government health facilities including one health centre and five dispensaries. Meanwhile, on each small island, population is mainly distributed between two main settlements: Kamarach and Kongata on Takawiri Island, Kibuogi A and Kibuogi B on Kibuogi Island, and Bonde and Luanda on Ngodhe Island. Takawiri and Kibuogi are close to Mfangano while Ngodhe is situated to the north of Rusinga Island. Takawiri and Ngodhe are each served by a dispensary, but no public health facility is available on Kibuogi. Ungoye, a small village on the mainland was included for comparative purposes due to its similarity to the islands in environmental characteristics, infrastructure, and access to the health facilities. It is served by a government dispensary. The village is connected by an unpaved road to the nearby towns of Sindo and Mbita, each with a sub-district hospital.This study was carried out in full accordance with all international, Kenyan and Swedish accepted guidelines as written informed consent was obtained from all participants. The Ethics Committee reviewed and approved all the consent procedures. This study was approved by the Kenyatta National Hospital/University of Nairobi-Ethics and Research Committee in Kenya (No.P7/1/2012) and the Committee on the Ethics of Human Research of Karolinska Institutet in Sweden (Dnr 201271239–31/4).Two cross-sectional surveys were performed in January and August 2012, after the short and long rainy seasons, respectively. Both surveys were conducted approximately 2 months after the rainy seasons to coincide with the periods of heaviest malaria burden. In our effort to ensure a high coverage of sampled population, each survey was conducted on different parts of the five study settings. The convenience sampling strategy was used in this study, whereby residents were asked to come to selected survey points such as community-based beach management unit (BMU) halls or school for study participation. Island and village leaders were sensitised to study by trained field workers and together provided information to community members at community meetings. Adult community members willing to participate were asked to read and provide signature on an informed consent form. In case of illiteracy, consent by thumb print was obtained in the presence of an independent literate adult witness. For children ≤12 years of age, consent was obtained from parents or guardians.21. On each small island, surveys were conducted separately between the two main settlements: Kamarach, Kibuogi A and Bonde in January and Kongata, Kibuogi B and Luanda in August. All surveys on small islands were conducted at BMU halls, which are typically connected to marketplaces.Different catchment areas were targeted in each survey Fig. . On MfanTo ensure a balanced representation of all age groups, five age categories were defined per study area: one to five years, six to ten years, 11–15 years, 16–30 years, and >30 years. Age and gender of each participant were recorded. Axillary body temperature was determined using a digital thermometer , and those with temperature exceeding 37.5 °C were considered febrile. Haemoglobin level was measured with the HemoCue Hb 201 Analyzer . A measurement below 11 g/dL was classified as anaemic. Children aged 12 years and below were examined for enlarged spleen by AK according to Hackett’s method, regardless of fever or malaria status.P. falciparum parasite in 100 high-power fields by experienced microscopists as described previously21. Parasite density was determined by counting the number of asexual parasites against 200 leukocytes and assuming that there are 8,000 leukocytes per µl of blood. DNA was extracted from a quartered blood spot (17.5 µl) using the QIAamp Blood Mini Kit  according to the manufacturer’s instructions. The presence of P. falciparum DNA was assessed using a nested PCR protocol (nPCR) as described previously57.A blood sample was obtained by finger prick (approximately 10 µl) for thick and thin blood smears, and two spots of blood (70 µl each) were collected on Whatman ET31 Chr filter paper . Blood spots on filter paper were air-dried and stored in plastic bags at 4 °C short term and at −20 °C for longer term. Blood smears were stained with Giemsa solution and examined for the presence of 58. The reconstituted blood spot solution, equivalent to a 1/200 dilution of serum, was stored at 4 °C until used for antibody test.A 3 mm disk was punched from each dried blood spot and serum was eluted in reconstitution buffer in 0.5 ml deep well plates  as described previouslyP. falciparum malaria antigens namely apical membrane antigen-1  and merozoite surface protein-1 , as well as to the P. falciparum sporozoite antigen NANP5 repeat peptide circumsporozoite protein (CSP) . Briefly, antigens were coated on NUNC-Immuno plates (Sigma) at concentrations of 0.5 µg/ml and 1.0 µg/ml in coating buffer (50 µl per well) for recombinant blood-stage antigens (AMA-1 and MSP-119) and CSP, respectively. The plates were washed in PBS with 0.05% Tween 20 (PBS/T) and blocked using 1% (w/v) skimmed milk solution  in PBS/T for three hours. After washing, 50 µl of reconstituted blood spot solution were added in duplicate at final dilutions of 1:1000 for both MSP-119 and CSP, and 1:2000 for AMA-1 and incubated overnight at 4 °C. A positive control consisting of a pool of hyper-immune sera was included in each plate. The plates were washed and 50 µl of horse-radish peroxidase (HRP)-conjugated rabbit anti-human IgG antibody  were added to all wells at a dilution 1:15,000 in PBS/T and incubated for three hours. After further series of washes, 100 µl of the substrate solution 3,3′,5,5′-tetramethylbenzidine (TMB)  were added. Reactions were stopped after 15 minutes with 50 µl per well of 0.2 M H2SO4. The optical density (OD) at 450 nm was read using Multiskan Go ELISA reader .All sera were tested for IgG antibodies by indirect quantitative enzyme-linked immunosorbent assay (ELISA) to two recombinant blood-stage 58. Seropositivity was determined by fitting a mixture model to normalised OD values assuming two Gaussian distributions, one for sero-negative individuals and another for sero-positive individuals25. The mean OD plus three standard deviations associated with the sero-negative group was used as the cut-off value for seropositivity. A separate cut-off was generated for each antigen. Differences in parasite prevalence and seroprevalence estimates between study areas and age categories were performed using a two-sided test for proportions and the corresponding exact binomial 95% confidence intervals (95% CIs). The titre of antibody responses was estimated using the equation dilution/[maximum OD/(OD test serum – minimum OD) – 1]; the median titre and interquartile range (IQR) are given. Differences in antibody responses were assessed using age-adjusted linear regression. Seroprevalence was stratified into yearly age groups and then analysed using a reverse catalytic modelling approach under a binomial sampling assumption, as described elsewhere13. This method provides estimates of the mean annual rates of conversion to seropositive  and reversion to seronegative  status, averaged over the age of the population. The common SRR was estimated from the model using maximum likelihood as described previously9. Infant under 1 year of age were excluded to remove any influence of maternally derived antibodies9. The serologically-derived annual EIR was estimated using AMA-1 and MSP-119 seroconversion rates and a calibration curve derived from determined values10. Factors associated with P. falciparum seropositivity were determined for each site separately using generalised estimating equations adjusting for correlation between observations from the same variables. The following factors were included: age in years, gender, fever, anaemia and enlarged spleen (children ≤12 years old). Variables that were significant at P < 0.10 in the univariate analyses were added to the multivariate model and retained in the final multivariate model if their association with immune responses was statistically significant at P < 0.05.Statistical analysis was performed using STATA/SE version 13.1  and GraphPad Prism Software version 5.03 . Duplicate ELISA OD values were averaged and normalised against values from blank wells as previously described to adjust for background reactivityThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.Supplementary Figure S1