hypertension is one of the most common if not the commonest medical complication in pregnancy.19 hyper - tensive disorders in pregnancy are found to be the greatest single cause of maternal mortality.911 it also causes a lot of prenatal mortality.12 most of the complications caused by this problem could be reduced by early detection and proper management.1314 studies on the incidence of hypertensive disease in pregnancy in most developing countries , including saudi arabia , are scarce.15 few of those studies , if any , have looked at the extent of knowledge , attitude or practice of doctors or nurses dealing with hypertensive pregnant mothers .
hence , the objective of this study was to determine the status of knowledge , attitude and practice ( kap ) of doctors and nurses in phc centers with regard to hypertension in pregnancy and to analyze factors affecting kap in the al - khobar area of the eastern province of the kingdom of saudi arabia .
terms used to describe hypertension and its complications in pregnancy differ according to its presentation , gestational age of discovery and the presence of previous history of hypertension before pregnancy .
the one defined by the 1972 committee on terminology of the american obstetricians and gynecologists ( acog)131519 was adopted for the purpose of this kap study .
doctors and female nurses working at all 8 phc centers in al - khobar area were enrolled for the study .
two self - administered questionnaires structured to examine the essential knowledge , attitude , and practice of doctors and nurses in dealing with hypertensive disorders during pregnancy .
the first was intended to gather demographic characteristics and information on the respondent that might affect the kap .
these variables included age , sex , nationality , year of graduation , certification , training and experience in obstetrics , duration of work in phc in the country and any in - service training received .
the second part of the questionnaire consisted of 49 questions divided into three sections dealing with practice , attitude and knowledge .
the practice sections included questions designed to test competence of doctors and nurses in the skills of measuring blood pressure , management of the hypertensive pregnant mother and practices of health education .
the knowledge section was composed of 28 questions to test basic medical knowledge regarding blood pressure reading , management of the problem and its complications .
both questionnaires were given to three consultant obstetricians and two nurse educators for consent validity and elimination of non - essential questions .
both knowledge and practice areas were scored on a zero and one additive scoring system in which each correct answer or practice was given one score and no mark given for a wrong response .
the attitude part included questions that measured attitudes of participants towards the seriousness and commonness of hypertension in pregnancy and their motivation to improve their knowledge on the subject .
attitude questions were scored using the five - point likert scaling system . in this scale ,
a high attitude was assigned to the answer if the respondent 's answer was to  agree  or
strongly agree  to the question in the scale and a low attitude level for  not sure  or
final scoring of the knowledge and practice section was satisfactory if the participant scored at least 60% of the total marks in these two sections .
this was an arbitrary cut - off point based on the pilot study and judgment of the experts . on attitude
, the questionnaire was handed over to five postgraduate doctors in general practice and their responses to attitude questions were used as a reference standard . the questionnaire for nurses
both versions of the questionnaire were pilot tested on a pilot group of 10 doctors and 22 nurses , selected randomly from phc centers in a nearby city , al - dammam to assess level of difficulty , clarity , suitability and time required for their completion .
data were fed into a personal computer , cleaned and analyzed using epi info and spss statistical packages .
frequency distribution tables were constructed and appropriate tests including the multiple regression analysis to identify significant independent factors were applied .
the total number of phc doctors in al - khobar area was 44 . among those 36 ( 81.4% ) who were present at time of data collection
their ages ranged between 25 - 47 with mean of 37.1 years ( sd  6.4 ) and 56% were in the 35 - 44 years group .
arab nationals other than saudis including egyptians , palestinian , and jordanian formed the majority of doctors ( 41.7% ) , while saudis constituted 22.2% of the group and the rest were from the indian subcontinent . the mean number of years in practice after graduation was 12.6 years ( sd  7.38 ) .
distribution of phc doctors according to years of practice after graduation the mean number of years working in phc was 4.7 years ( sd  3.4 ) and 17 doctors ( 47.2% ) had worked for over 5 years in phc centers . only , 4 doctors ( 11.1% ) had postgraduate qualifications ; out of these only one held a diploma in obstetrics .
five doctors ( 13.9% ) had had 3 - 6 months post - internship training in obstetrics but no certification .
of the entire group , only 2 doctors ( 5.6% ) had had courses in hypertensive disorders in pregnancy in the course of their work . in describing their general attitude towards managing hypertension in pregnancy ,
a majority of doctors ( 80% ) stated clearly that they did not feel confident in managing hypertensive pregnant mothers and would prefer to see children or adult male patients in their clinic . nevertheless , the attitude of 34 doctors ( 94.4% ) toward learning more about hypertension in pregnancy was positive .
out of the maximum attitude score of 45 marks , the mean score for the attitude questions for the group reached a reasonably high figure of 30.69 marks .
doctors attitudes towards hypertension in pregnancy doctors management of hypertension in pregnancy was quite deficient as 16 doctors ( 44.4% ) reported that they had not actually seen any hypertensive pregnant mother during last year .
around 53% of the doctors depended on bp measurement taken by a competent staff and did not check it .
the doctors scores for practice was generally low , as only 60% of them got the correct answers to the skill of blood pressure measurement , a basic skill necessary for any medical graduate .
in addition , almost all doctors wrongly reported that they had advised their pregnant hypertensive patients to lose weight and reduce their salt intake .
another major mistake noted among 25% of the doctors was that they said they would start treatment of hypertensive cases immediately after diagnosis in their clinic without considering referral .
the scores on practice of 75% of the doctors were below the satisfactory cut - of point .
again the doctors score of 58.3% on all areas of knowledge was unsatisfactory since this directly affected the diagnosis and quality of care for hypertensive mothers .
some of the responses dealt with normal and abnormal readings of blood pressure and the presenting symptoms of preeclampsia .
doctors mean score 12.1 marks out of a total score of 21 on knowledge was moderate .
the total number of phc nurses in al - khobar area was 120 . among these ,
the 91 ( 75.8% ) who were present at the time of data collection answered the questionnaire .
their mean age was 29.8 years ( sd  6.3 ) ranging between 20 - 44 years , 74% of them fell within the 20 - 34 age group .
indians formed the second largest group ( 37.4% ) followed by filipinos ( 11% ) . according to their qualification , 14 nurses ( 15.4% ) held bachelor degrees and 77 ( 84.6% ) had diplomas .
mean years of practice after registration was 8.7 ( sd  5.4 ) and their distribution is shown in table 3 .
mean duration of work in phc was 4.1 years ( sd  2.8 ) and most nurses ( 69.2% ) had spent < 5 years working in phc in the country .
distribution of phc nurses according to years of practice after registration thirty - seven nurses ( 40.7% ) had spent < 6 months in obstetrics training and 10 nurses ( 11% ) had spent > 1 year .
most of the group ( 48.3% ) , mainly the saudis , had minimal training of 3 weeks in obstetrics ward during their internship period as required by the saudi female health institution .
a majority of the nurses  73 ( 80.2% ) had not worked in a phc setting before their current positions . out of those who had had previous phc experience ,
nine of them had had courses in hypertensive disorders in pregnancy during their work in phc . in describing their general attitude towards hypertension in pregnancy
, 86% of them reported that it was a common health problem and they were all positive about learning more .
they were all keen to talk to pregnant women about their problem and advice them to take bed rest .
out of the maximum attitude score of 35 marks , the highest mean score was 30.02 marks . table 4 shows some of the attitude questions and their responses . nurses attitude towards hypertension in pregnancy
the nurses were quite good on practice in the area of hypertension in pregnancy , as 95.6% of them scored  satisfactory  .
the mean score of this section on practice was quite high for nurses being 8.3 marks out of a total score of 12 marks .
the nurses level of knowledge was very low since only five of them ( 5.5% ) scored  satisfactory  on the questions in this section .
the deficiencies were in areas dealing with diagnosis and quality of care for hypertensive mothers .
most of them ( 96.7% ) wrongly considered salt restriction and weight reduction as important in the health education of hypertensive pregnant women .
mean scores on knowledge was 9.02 marks out of a total score of 20 marks which was quite low . in the regression analysis of independent factors associated with kap of doctors and nurses , all variables in the first part of the questionnaire such as age , nationality and certification
were included in a series of multiple regression equations against each of the dependent variables , namely , scores on knowledge , attitude , and practice for doctors and nurses .
there was a positive significant association of the doctors attitude with their sex i.e. , male , nationality , being older and previous enrollment in a course on hypertension ( r square = 0.26 p=0.02 ) .
their knowledge was only associated with previous training in obstetrics ( r square = 0.14 , p=0.02 ) , while practices of both doctors and nurses were not associated with any of the factors under study .
the attitude of nurses , on the other hand , showed different associations as non - saudi nationals and training in obstetrics had a positive significant effect on their attitude ( r square = 0.08 , p=0.02 ) .
their knowledge was only associated significantly with being non - saudi ( r square = 0.19 , p=0.001 ) .
hypertension in pregnancy is one of the major causes of prenatal morality and morbidity.20 it is responsible for about 18% of maternal mortality,102122 maternal mortality in hypertensive disorders of pregnancy is primarily due to low standard of care and delay in referral.14 antenatal care ( anc ) is a major part of maternal and child services in phc .
one of the most important functions of anc is to detect high - risk pregnancies and to give them the necessary care .
findings from this study will help to identify the status kap of workers in phc on hypertensive disorders in pregnancy .
it was discovered in this study that the definition of raised blood pressure and its management was not standardized among doctors and nurses in phc .
this is not surprising as previous study by bisson in bristol23 in which a large group of general practitioners , hospital doctors , hospital midwives , community midwives and student midwives who were questioned , gave variety of action plans according to their understanding of diagnostic criteria .
they considered the reading of 90 mmhg the model value of diastolic bp at which further action would be taken , whether proteinuria was present or absent .
edema was considered a useful indicator by 93% of the respondents and 49% would use ankle edema in their assessment .
another questionnaire - based survey by hutton24 on the management of hypertension in pregnancy completed by 65 new zealand obstetricians found that 40 ( 61.5% ) doctors considered the diastolic of 80 - 85 mmhg , the lowest abnormal reading , at 28 weeks , and by 18 ( 27.7% ) doctors at 36-week gestation . however , 20 ( 30.8% ) doctors considered the diastolic of 90 mmhg , at 28 weeks and 42 ( 64.6% ) doctors at 36 weeks gestation , the lowest abnormal reading . around 47% of phc doctors
had spent more than 5 years in phc service in ksa and only 14% of them had had post - internship training in obstetrics but being males had not been involved in anc activity in phc . only 2 had had courses in hypertensive disorders of pregnancy .
about 44% of the doctors had not seen any cases of hypertensive pregnant mothers during the last year .
questions asked were concerned with four aspects namely , techniques of blood pressure measurements , history and physical examination , health education practice and action management to be taken by the phc doctors on discovered cases .
it is obvious that having such little contact with hypertensive cases and lacking the basic training or refresher courses , their management skills will be inadequate .
since hypertension is one of the commonest medical complications in pregnancy , it is important to educate the pregnant mother about it .
almost all doctors report that they advise hypertensive mothers on the need for bed rest .
this is an important non - pharmacological measure in the management of the problem.25 on the other hand , almost all doctors report that they advise their patients to restrict their salt intake and to go on a weight - reducing diet .
salt restriction in hypertensive non - pregnant women can be effective but in pregnancy may aggravate the condition .
moreover , diet restriction in pregnancy can lead to delivery of small - for - date fetus.25 similar findings of wrong advice were reported in other studies by trudinger26 and bisson.23 around 85% of phc doctors reported that they would not refer hypertensive cases after diagnosis and 25% of them would start medication immediately after diagnosis .
in fact , the cornerstone of management of hypertensive cases starts in the phc center with accurate diagnosis and undelayed referral of these cases from the phc to the specialist or hospital.1621 phc doctors should be aware of their limitation in the management of these cases and not to jeopardize the health of the mother and her fetus .
around 31% did not recognize this is a common health problem in pregnancy , while 97% of them stressed the importance of taking blood pressure reading at each anc visit .
that 53% of phc doctors depended on blood pressure measurement taken by clinical staff without confirming it , the reading themselves is an alarming negative attitude as well as wrong practice .
the diagnosis of hypertension in general or in pregnancy in any patient is not an easy task and proper management and modifications depend on it .
it is therefore , important that the person responsible for the management , namely , the doctor , should confirm the reading again . in looking for factors affecting the attitude of doctors
, it was found that gender i.e. , male , arab , older and previous training in a course on hypertension were factors associated with higher scores . longer experience in phc work , being male and older and the absence of a language barrier for arabic speakers , including saudis , might explain to some extent their better attitude scores as compared to those of the females and younger doctors .
the knowledge of about 58% of doctors on questions that bare on the diagnosis and quality of care of hypertensive mothers as well as
nurses provide most of the vital anc services in the phc . around 53% of the phc nurses
were non - saudis and non - arabic speakers and the language barrier adversely affects the health education in hypertensive cases .
it is obvious from the results that there is a shortage of specialized courses for nurses .
none of the saudi nurses had more than a diploma in nursing and there did nt seem to be any arrangements for their further movement at universities .
a period of 3 weeks in an obstetrics ward for new graduate nurses as part of an internship period is not enough to give clear , pragmatic information on anc services in a phc setting .
about 31% of the nurses have spent > 5 years in the phc centers in the ksa , but only 10% had taken courses in hypertension .
this clearly , demonstrates the necessity of a better arrangement for on - job training classes for them .
their responses to the question of history and physical examinations were 87% correct and about 90% of them responded correctly to the questions on health education .
their erroneous responses were on the items on restriction on salt intake and weight reduction .
these good scores indicate that except for their wrong ideas on salt and diet restriction , which should be corrected at refresher courses , the correct procedure have been learnt . although the phc nurses were very good with practice in general , around 95% of them had poor knowledge .
knowledge and facts , on the other hand , need to be updated by continuous education .
around 86% of them felt that it was a common health problem and reported that they needed to know more about it .
the importance of a health education as a vital task for nurses can not be ignored .
the presence of these cases in the clinic , therefore , provides a good opportunity for the performance of this task .
there was a strong positive association of attitude of nurses to non - saudi nationality and training in obstetrics .
while all saudi nurses hold diplomas , about 30% of the non - saudi nurses have bachelors degrees .
there is a masked difference in the type , duration and content of curriculum at the pre - graduate level for both groups and non - saudi nurses had higher knowledge scores than saudis .
the implication of these findings clearly points out the need to improve the knowledge and attitude of phc nurses through refresher courses .
good  in attitude questions but had low scores on the practice and knowledge component of the questionnaire .
the nurses scored high on practice and attitude but had low scores on the knowledge component .
it is recommended that appropriate regular refresher courses on common and serious problems like hypertension be organized for doctors and nurses in the phcs .
there should be opportunities for effective training of reasonable duration with clearly defined objectives under proper supervision in good hospitals to improve their knowledge and practice
. it would be also appropriate to offer saudi doctors extra incentives for postgraduate study in family medicine to deal with these common problems , and to institute a suitable program of continuing medical education within the health centers for both doctors and nurses .
it is also vital to review the curriculum of the female nursing institutions to update both its theoretical and practical content , and extend the duration of training in such common problems as hypertension in obstetrics .

 objective : to assess the status of knowledge , attitude and practice ( kap ) of doctors and nurses in primary health care ( phc ) centers with regard to hypertension in pregnancy and to identify factors associated with kap in al - khobar , saudi arabia.methodology:using a self - administered comprehensive questionnaire , all available doctors and nurses in phc centers of the al - khobar area were approached to determine their knowledge , attitude and practice in hypertension during pregnancy .
 questionnaires were validated and pilot tested .
 each section of the questionnaire was scored and the mean scores calculated .
 factors affecting each section were identified by means of multiple regression analysis.results:a total of 36 doctors and 91 nurses were enrolled in the study .
 saudis formed 22.2% of the doctors and 47.3% of nurses .
 mean years of practice after graduation were 12.6 and 8.7 years for doctors and nurses , respectively .
 saudi nurses spend only 3 weeks in the obstetrics ward during the whole period of their internship .
 all saudi nurses hold only diplomas and not many courses on the hypertensive disorder are offered to both doctors and nurses after graduation .
 the practice of doctors particularly in the management of patients after reaching a diagnosis and educating them on diet and salt intake was poor .
 furthermore , their knowledge was also poor . though their level of knowledge was poor , the nurses practice was satisfactory , particularly in taking history and physical examination
 .
 the attitude of both doctors and nurses towards hypertensive disorders was in general , positive and satisfactory towards health education .
 nurses nationality and duration of post - internship training were the factors that influenced their attitude and scores on knowledge.conclusion and recommendation : the study revealed that both doctors and nurses working in the phc lacked training and knowledge in this area of their work .
 it is therefore necessary to give phc doctors and nurses refresher courses on common and serious problems like hypertension . a longer period of training in action management is needed to improve the knowledge and practice of doctors and nurses working in antenatal clinics in this area .


renal cell carcinoma ( rcc ) is the most common primary malignant neoplasm of the kidney and accounts for about 3% of all adult neoplasms .
the incidence of rcc has been increasing during the past two decades in all age groups .
the greatest increase has been in patients with localized tumors as a result of incidental detection , which is thought to be the result of more widespread use of abdominal ultrasound and computed tomography . however , about 30% of patients with rcc already have metastatic disease at the time of diagnosis , and 10% to 14% of patients experience local recurrence or distant metastasis after nephrectomy despite a finding of pathologically confined disease at the time of nephrectomy [ 3 - 5 ] .
well - known pathological and clinical variables have been identified and sometimes integrated into nomograms to better predict disease recurrence , but with only partial success . with a better understanding of the biology of rcc
, laboratory values and molecular markers are increasingly being investigated as potential predictive factors for rcc [ 6 - 8 ] .
disease progression appears to depend on a complex interaction between the tumor and host inflammatory responses .
it is estimated that underlying infection and inflammatory responses are linked to 15% to 20% of all deaths from cancer worldwide .
the serum level of c - reactive protein ( crp ) , a nonspecific inflammatory acute - phase protein , is frequently increased in patients with metastatic rcc and is a predictor of prognosis .
however , secondary thrombocytosis has been reported with many malignant diseases ; the survival of such patients has been reported to be significantly shorter . the prognostic value of the platelet count has been studied in patients with localized and metastatic rcc ; patients with thrombocytosis had a poorer survival than did patients with a normal platelet count . the purpose of this study was to investigate the association of preoperative crp elevation and thrombocytosis with the prognosis of patients with non - metastatic rcc .
the medical records of 177 patients with non - metastatic rcc who underwent a radical nephrectomy between march 2000 and may 2008 and for whom preoperative crp and platelet levels were available for analysis were retrospectively reviewed .
there were 130 men and 43 women , with a mean age of 53.5 years ( range , 28 - 83 years ) .
preoperatively , all patients were evaluated with a physical examination , routine hematology and biochemical analysis , and radiology studies , including abdominal computed tomography and chest x - ray .
the tumors were staged by using the 2002 tnm classification of the american joint committee on cancer ( ajcc )   and were graded according to fuhrman 's nuclear grading system .
all patients were evaluated postoperatively every 3 months for the first 2 years , every 6 months for the next 2 years , and yearly thereafter .
crp elevation was defined as a crp level 0.8 mg / dl , and thrombocytosis was defined as a platelet count 380,000/l in men and 370,000/l in women according to the normal reference range in our hospital .
disease recurrence was defined as local failure in the tumor bed or regional lymph nodes or distant metastasis .
the chi - square test or fisher 's exact test was used to analyze the correlation between preoperative crp elevation or thrombocytosis and the clinical and pathological variables , including age , gender , anemia , histology subtype , tumor size , t stage , nuclear grade , and metastasis during follow - up .
univariate and multivariate analyses were performed by using the log - rank test or the cox proportional hazards regression model . for all tests ,
the clinical and pathological characteristics of the 177 patients with non - metastatic rcc are summarized in table 1 .
the mean tumor size was 5.12 cm ( median , 4.50 ; range , 1 - 22 cm ) .
regional lymph node dissection was performed in 24 patients who showed suspicious lymph node metastasis on computed tomography . however , none had a pathological diagnosis of lymph node metastasis .
there were 38 patients ( 21.5% ) with crp elevations and 11 patients ( 6.2% ) with thrombocytosis . among patients with crp elevation ,
the mean follow - up period was 48.3 months ( median , 48.0 ; range , 13 - 111 months ) .
twenty - three patients ( 13.0% ) developed metastases and six patients died during the follow - up period .
preoperative crp elevation was significantly correlated with anemia ( p=0.001 ) , t stage ( p=0.004 ) , grade ( p=0.025 ) , and metastasis ( p<0.001 ) , but not with age , gender , tumor histology subtype , or tumor size ( table 2 ) .
preoperative thrombocytosis was significantly correlated with anemia ( p=0.003 ) , t stage ( p=0.002 ) , and metastasis ( p=0.001 ) , but not with age , gender , tumor histology subtype , tumor size , or grade ( table 3 ) .
no significant correlation was found between preoperative crp levels and platelet counts when analyzed by using pearson 's correlation coefficient ( r=0.132 , p=0.079 ) .
kaplan - meier recurrence - free survival curves according to crp level showed that the survival rate of patients with crp elevation ( crp0.8 mg / dl ) was significantly lower than that of patients with normal crp levels ( p<0.001 ) ( fig .
the univariate analysis identified anemia , crp elevation , thrombocytosis , tumor histology subtype , tumor size , t stage , and grade as significant prognostic factors for recurrence - free survival , whereas the multivariate analysis showed that crp elevation ( p=0.033 ) and tumor size ( p=0.007 ) were independent prognostic factors ( table 4 ) .
crp is an acute phase reactant that is produced exclusively by hepatocytes in response to cytokines such as interleukin ( il)-6 , which are known mediators of ongoing inflammatory processes .
an elevation of the serum concentration of this protein indicates the presence of acute inflammation , which is observed 8 to 12 hours after the onset of infection . the relationship between cancer and crp elevation
hepatic production of crp is induced by cytokines such as il-1 , tumor necrosis factor ( tnf ) , and primarily il-6 , which is frequently overproduced by the tumor cells themselves .
experimental studies on rcc cell lines and expression studies of renal surgical specimens have shown that at least some renal tumors produce il-6 , which functions as an autocrine growth factor of the rcc .
therefore , the presence of a systemic inflammatory response might be associated with aggressive rcc behavior .
the prognostic value of crp levels has been reported in many recent studies of cancers , including those of the esophagus , ovaries , and colon [ 21 - 23 ] .
however , the mechanism by which a systemic inflammatory response affects cancer - specific survival in patients with rcc is unclear .
hwang et al studied the relationship between crp and survival in patients with rcc who underwent a radical nephrectomy .
they reported a statistically insignificant difference in the survival rates of patients with and without crp elevation and concluded that crp was not a significant prognostic factor for rcc when compared with tumor stage , grade , tumor size , and cell type .
however , komai et al demonstrated that the 5-year and 10-year disease - specific survival rates ( 75% and 30% , respectively ) in patients with high crp levels were significantly worse than the rates in patients with normal crp levels ( both 93% , p<0.001 ) and suggested that the preoperative crp level is associated with poor survival in patients with localized rcc .
in addition , they reported that the crp level is a useful preoperative screening tool in patients with localized rcc .
blay et al reported that pretreatment il-6 and crp were significantly associated with the effects of cytokine therapy and high il-6 and that the crp level indicated a poor response to cytokine therapy . in the present study ,
preoperative crp elevation was associated with a poorer prognosis and a higher recurrence rate in patients with non - metastatic rcc .
this result confirms the importance of the preoperative crp level as an independent prognostic factor for tumor recurrence and is consistent with the findings of earlier reports showing that a high crp level is associated with poor survival in localized rcc .
secondary or reactive thrombocytosis has been associated with tumor metastasis and poor survival in cancer patients .
the pathophysiologic mechanism of reactive thrombocytosis may be tumor - associated elevation of circulating platelets by tumor - derived humoral factors such as il-6 and macrophage colony - stimulating factor ( m - csf ) that may be responsible for the development of cancer - related thrombocytosis .
il-6 is a potent stimulator of megakaryocytopoiesis , and tumor cells have been shown to release il-6 both in vitro and in vivo .
these observations suggest that il-6 plays a role in the development of tumor - associated thrombocytosis .
karakiewicz et al analyzed 1828 patients with rcc by univariable , multivariable , and predictive accuracy analyses with regard to rcc - specific mortality .
in that study , they showed that the addition of thrombocytosis to the base model ( age , tumor size , tnm stage , ecog - performance status , fuhrman grade , and histology subtype ) increased the predictive accuracy by only 0.3% ( from 85.3% to 85.6% ) ; these changes in predictive accuracy were not statistically significant .
consequently , they concluded that patients who presented with thrombocytosis did not have poorer prognosis than did their counterparts who did not exhibit these apparently unfavorable characteristics , as long as the effects of the tnm stage , histology , tumor grade , and ecog - performance status were considered .
however , many investigators have reported that thrombocytosis is related to a poor prognosis in patients with rcc .
, they showed that patients with thrombocytosis had a mean survival of 45.2 months compared with 76.6 months in patients without thrombocytosis ( p=0.0002 ) and concluded that preoperative thrombocytosis is associated with a poorer survival than is a normal platelet count . in the present study
, we showed that preoperative thrombocytosis was significantly correlated with anemia , t stage , and metastasis .
in addition , thrombocytosis was a significant prognostic factor associated with recurrence - free survival in the univariate analysis .
therefore , thrombocytosis was associated with a poorer prognosis and a higher recurrence rate in patients with non - metastatic rcc ; these findings are consistent with those of previous studies .
ito et al showed that preoperative crp levels and platelet counts had a significant correlation and suggested that this was because the reactive thrombocytosis and crp elevation in rcc were both caused by the production of inflammatory cytokines such as il-6 .
as in the study by ito et al , we analyzed the relationship between preoperative crp and platelet count .
however , we did not find a statistically significant association between preoperative crp and platelet count ( r=0.132 , p=0.079 ) .
therefore , the exact relationship between preoperative crp and platelet count remains to be determined .
a limitation of this study is that several variables could not be considered in the current analysis .
nonetheless , all available variables were included , and our findings indicate that several known prognostic factors were valuable as prognostic factors
the reason for this result was , to some extent , too many patients in stage t1 rather than other t stages and a small number of patients with grade 4 compared with other grades according to the early detection of rcc nowadays .
preoperative crp elevation and thrombocytosis were associated with a poorer prognosis and a higher recurrence rate in patients with non - metastatic rcc . moreover ,
however , preoperative thrombocytosis was not an independent prognostic factor for tumor recurrence and prognosis .

 purposethe aim of this study was to investigate the association of preoperative c - reactive protein ( crp ) elevation and thrombocytosis with the prognosis of patients with non - metastatic renal cell carcinoma ( rcc).materials and methodsthis was a retrospective review of the medical records of 177 patients ( 130 men and 47 women ) with non - metastatic rcc who underwent a radical nephrectomy between march 2000 and may 2008 and for whom preoperative crp and platelet data were available for analysis .
 preoperative crp elevation and thrombocytosis were compared with clinical and pathological variables.resultsthere were 38 patients with crp elevation and 11 patients with thrombocytosis .
 the mean follow - up time was 48.3 months ( median , 48.0 ; range , 13 - 111 months ) .
 twenty - three patients ( 13.0% ) developed metastases and six patients died during the follow - up period .
 crp elevation was significantly correlated with anemia ( p=0.001 ) , t stage ( p=0.004 ) , grade ( p=0.025 ) , and metastasis ( p<0.001 ) .
 thrombocytosis was significantly correlated with anemia ( p=0.003 ) , t stage ( p=0.002 ) , and metastasis ( p=0.001 ) .
 the univariate analysis identified anemia , crp elevation , thrombocytosis , tumor histology subtype , tumor size , t stage , and grade as significant prognostic factors associated with recurrence - free survival , whereas the multivariate analyses showed that crp elevation ( p=0.033 ) and tumor size ( p=0.007 ) were independent prognostic factors.conclusionspreoperative crp elevation and thrombocytosis were associated with a poorer prognosis and a higher recurrence rate in patients with non - metastatic rcc .
 moreover , preoperative crp elevation appeared to be an independent predictor of tumor recurrence and prognosis .
 preoperative thrombocytosis , however , was not an independent prognostic factor for tumor recurrence and prognosis .


angiogenesis is a constant hallmark of multiple myeloma ( mm ) progression and has prognostic potential .
we have previously isolated endothelial cells ( ec ) from bone marrow of patients with mm ( mmec ) .
they show intrinsic angiogenic ability , because they rapidly form a capillary network in vitro , and extrinsic ability , because they generate numerous new vessels in vivo in the chick embryo chorioallantoic membrane ( cam ) .
moreover , mmec secrete angiogenic cytokines , such as fibroblast growth factor-2 ( fgf-2 ) and vascular endothelial growth factor ( vegf ) , as well as matrix metalloproteinase-2 and 9 ( mmp-2 and mmp-9 ) and express adhesion molecules facilitating dissemination of these cells .
studies by rajkumar et al . , dominici et al . , and laroche et al .
the bone marrow microenviromnent plays a crucial role in inducing the angiogenic response in mm [ 6 , 7 ] .
reciprocal positive and negative interactions between plasma cells and bone marrow stromal cells , namely haematopoietic stem cells , fibroblasts , ostoblasts / osteoclasts , ec , ec progenitor cell , t lymphocytes , macrophages and mast cells are mediated by an array of cytokines , receptors and adhesion molecules .
these cells secrete angiogenic factors , such as interleukin-6 ( il-6 ) , vegf , fgf-2 , hepato - cyte growth factor / scatter factor ( hgf / sf ) , transforming growth factor- ( tgf- ) and tumour necrosis factor  ( tnf- ) [ 6 , 7 ] . here , we have attempted a fine characterization of the angiogenic response induced by mmec , as compared with monoclonal gammopathy of undetermined significance ec ( mgusec ) in the cam assay .
we investigated the angiogenic response induced by gelatin sponges soaked with the cell suspensions and implantedonthecamsurfacefrom8today12ofincu - bation and we studied the effects of mmec and mgusec on the expression of endogenous levels in the cam of vegf , fgf-2 , angiopoietin-1(ang-1 ) , hypoxia - inducible factor-1 ( hif-1 ) and endostatin by reverse transcriptase - polymerase chain reaction ( rt - pcr ) .
mmec were obtained from 10 patients with mm and mgsuec were obtained from 10 patients with mgus , as previously described .
fertilized white leghorn chicken eggs were incubated at 37c at constant humidity . on day 3 of incubation a square window was opened in the egg shell after removal of 23 ml of albumen so as to detach the developing cam from the shell .
the window was sealed with a glass and the eggs were returned to the incubator . gelatin sponges ( gelfoam , upjohn company , kalamazoo , mi , usa ) were cut to a size of 1 mm3 and placed on top of a growing cam at day 8 incubation under sterile conditions .
the sponges were then adsorbed with 2 l of cell suspension ( 18,000 cells per sponge ) of mmec or mgusec or with rpmi-1640 medium alone or supplemented with fgf-2 ( 200 g / ml ) ( r & d systems , abington , uk ) , used as negative and positive control , respectively .
the angiogenic response was evaluated 96 hrs after the implants by means of a stereomicroscope connected to an image analyser system ( olympus italia , italy ) .
blood vessels entering the sponges within the focal plane of the cam were counted by two observers in a double blind fashion at a magnification of 50x .
means  1 standard deviation ( sd ) were evaluated for all the parameters and the statistical significance of the differences between the counts was determined by student 's t - test for unpaired data .
total rna was extracted from 12 cam treated with ec obtained from six patients with mgus and six patients with mm and from six control cam using trizol reagent ( invitrogen , carlsbad , ca , usa ) according to the manufactures 's protocol . the concentration and the purity of the isolated rna
were determined using a spettrofotometric analysis and the integrity of the rna was verified by means of electroforesis in a formaldehyde agarose gel followed by ethidium bromide staining .
the cdna was then synthetized with 5 g of total rna using m - mlv and oligo ( dt)1820 as primer ( invitrogen , carlsbad , ca , usa ) .
level of mrna expression of pro - angiogenic molecules vegf , fgf-2 , ang-1 , hif-1 and mrna expression of anti - angiogenic factor endostatin were evaluated by pcr using specific primers and the relative level of the each amplified transcripts were normalized to those of the housekeeping gene -actin .
pcr reactions were performed in a final volume of 25 l containing 2 l of rt products , 10 picomoles of each primer and 1x of red - taq polymerase mix ( sigma - aldrich , milano , italy ) .
preliminary experiments were performed to determine the appropriate conditions ( ta and specific number of cycles ) for semi - quantitative rt - pcr and transcript were amplified using different cycle numbers:25 cycles for -actin , 30 cycles for vegf , fgf-2 , ang-1 and for endostatin and 35 cycles for hif-1. pcr products were subjected to electrophoresis in a 1.5% agarose gel and ethidium bromide stained , and then photographed under an ultraviolet transilluminator ( amersham biosciences ) .
intensity of bands was quantified as arbitrary optical density units ( od ) using the scion image software ( based on nih image ) .
six cam treated with ec obtained from three patients with mgus and three patients with mm and three control cam were homogenized in ice cold buffer containing 250 mm sucrose , 10 mm tris - hcl , ph 7.4 and protease inhibitors [ 1g / ml leupeptin , 1g / ml pepstatin and 1 mm phenyl - methylsulfonyl fluoride ( pmsf ) ] .
after homogenization in a potter apparatus and centrifugation at 1000 g for 10 min , the protein content of the supernatant was measured using a bradford assay ( bio - rad laboratories , hercules , ca , usa ) . for sds - page , samples ( 60 g / lane ) were solubilized in laemmli buffer , boiled at 90c for 10 min and runned on a 12% polyacrylamide .
the proteins were blotted to a pvdf membrane ( millipore corporate , billeira , ma , usa ) and immunoblotting was performed using a monoclonal mouse primary antibody against endostatin ( novus biologicals , littleton , co , usa ) diluted 1:500 in tbst .
peroxidase activity was revealed by chemiluminescence ( ecl kit , santa cruz biotechnology , santa cruz , ca , usa ) and blot was immediately exposed to x - ray film .
control experiments were performed using a mouse - anti--actin ( abcam , cambridge , ca , usa ) to detect housekeeping protein -actin to normalize the protein load .
stained bands were scanned and intensity was quantified using the scion image system ( based on nih image ) .
mmec were obtained from 10 patients with mm and mgsuec were obtained from 10 patients with mgus , as previously described .
fertilized white leghorn chicken eggs were incubated at 37c at constant humidity . on day 3 of incubation a square window was opened in the egg shell after removal of 23 ml of albumen so as to detach the developing cam from the shell .
the window was sealed with a glass and the eggs were returned to the incubator .
gelatin sponges ( gelfoam , upjohn company , kalamazoo , mi , usa ) were cut to a size of 1 mm3 and placed on top of a growing cam at day 8 incubation under sterile conditions .
the sponges were then adsorbed with 2 l of cell suspension ( 18,000 cells per sponge ) of mmec or mgusec or with rpmi-1640 medium alone or supplemented with fgf-2 ( 200 g / ml ) ( r & d systems , abington , uk ) , used as negative and positive control , respectively .
the angiogenic response was evaluated 96 hrs after the implants by means of a stereomicroscope connected to an image analyser system ( olympus italia , italy ) .
blood vessels entering the sponges within the focal plane of the cam were counted by two observers in a double blind fashion at a magnification of 50x .
means  1 standard deviation ( sd ) were evaluated for all the parameters and the statistical significance of the differences between the counts was determined by student 's t - test for unpaired data .
total rna was extracted from 12 cam treated with ec obtained from six patients with mgus and six patients with mm and from six control cam using trizol reagent ( invitrogen , carlsbad , ca , usa ) according to the manufactures 's protocol . the concentration and the purity of the isolated rna
were determined using a spettrofotometric analysis and the integrity of the rna was verified by means of electroforesis in a formaldehyde agarose gel followed by ethidium bromide staining .
the cdna was then synthetized with 5 g of total rna using m - mlv and oligo ( dt)1820 as primer ( invitrogen , carlsbad , ca , usa ) .
level of mrna expression of pro - angiogenic molecules vegf , fgf-2 , ang-1 , hif-1 and mrna expression of anti - angiogenic factor endostatin were evaluated by pcr using specific primers and the relative level of the each amplified transcripts were normalized to those of the housekeeping gene -actin .
pcr reactions were performed in a final volume of 25 l containing 2 l of rt products , 10 picomoles of each primer and 1x of red - taq polymerase mix ( sigma - aldrich , milano , italy ) .
preliminary experiments were performed to determine the appropriate conditions ( ta and specific number of cycles ) for semi - quantitative rt - pcr and transcript were amplified using different cycle numbers:25 cycles for -actin , 30 cycles for vegf , fgf-2 , ang-1 and for endostatin and 35 cycles for hif-1. pcr products were subjected to electrophoresis in a 1.5% agarose gel and ethidium bromide stained , and then photographed under an ultraviolet transilluminator ( amersham biosciences ) .
intensity of bands was quantified as arbitrary optical density units ( od ) using the scion image software ( based on nih image ) .
six cam treated with ec obtained from three patients with mgus and three patients with mm and three control cam were homogenized in ice cold buffer containing 250 mm sucrose , 10 mm tris - hcl , ph 7.4 and protease inhibitors [ 1g / ml leupeptin , 1g / ml pepstatin and 1 mm phenyl - methylsulfonyl fluoride ( pmsf ) ] .
after homogenization in a potter apparatus and centrifugation at 1000 g for 10 min , the protein content of the supernatant was measured using a bradford assay ( bio - rad laboratories , hercules , ca , usa ) . for sds - page , samples ( 60 g / lane ) were solubilized in laemmli buffer , boiled at 90c for 10 min and runned on a 12% polyacrylamide .
the proteins were blotted to a pvdf membrane ( millipore corporate , billeira , ma , usa ) and immunoblotting was performed using a monoclonal mouse primary antibody against endostatin ( novus biologicals , littleton , co , usa ) diluted 1:500 in tbst .
peroxidase activity was revealed by chemiluminescence ( ecl kit , santa cruz biotechnology , santa cruz , ca , usa ) and blot was immediately exposed to x - ray film .
control experiments were performed using a mouse - anti--actin ( abcam , cambridge , ca , usa ) to detect housekeeping protein -actin to normalize the protein load .
stained bands were scanned and intensity was quantified using the scion image system ( based on nih image ) .
the ability of mmec and mgusec to induce an angiogenic response in vivo was assessed with the cam - gelatin sponge assay .
1 ) . cam implanted with the medium containing fgf-2 or mmec gave significantly higher vessel counts and numerous allantoic vessels converging like spokes toward the sponges were recognizable ( table 1 and fig .
when the sponges were loaded with the medium alone and with mgusec , physiologic angiogenesis was observed in the form of few allantoic vessels partly around and partly converging toward the sponge ( table 1 and fig .
angiogenic response assessed by the chick embryo chorioallantoic membrane ( cam)-gelatin sponge assay p<0.001 versus rpmi-1640 and mgusec angiogenic activity in the chorioallantoic membrane ( cam ) assay . gelatin sponges loaded with fgf-2 ( a ) , mmec ( b ) , rpmi-1640 ( c ) and mgusec ( d ) were implanted on top of the cam on day 8 .
macroscopic view of the cam on day 12 shows numerous allantoic vessels converging like spokes toward the sponge in a and b , while few allantoic vessels are recognizable in c and d. original magnification : a  d , x 50 . in order to evaluate the angiogenic effects of mmec and mgusec , mrna expression of pro - angiogenic factors including vegf , fgf2 , ang-1 , hif-1 and mrna expression of anti - angiogenic molecule endostatin
the expression level of each transcript was compared to those detected in control cam . as shown in fig .
2 , treatment of cam with mmec and mgusec did not produce significant difference in the expression of pro - angiogenic factors mrna in comparison with control cam treated with medium alone .
moreover , no statistical difference was found in vegf , fgf-2 , ang-1 , hif-1 and endostatin mrna content between mm and mgus treated cam ( p<0.05 ) .
in contrast , the expression of endostatin mrna detected in both mgus and mm treated cam was significantly lower respect to control cam ( mgus od:120.113 and mm od:48.319.6 versus ctrl od:205.84  19.6 ; p<0.05 ) .
expression levels of mrna coding for vegf , fgf-2 , ang-1 , hif1 and endostatin evaluated by semi - quantitative rt - pcr .
transcript levels from the cam assay are referred to ec obtained from 6 mm and 6 mmgus patients and the error band represents the standard deviation of six experiments .
western blot analyses were also performed to study whether the expression of endostatin changes in mmec and mgusec treated cam respect to control . as shown in fig .
3 , according to rt - pcr quantification , endostatin was significantly lower also at the protein level in both mgusec and mmec treated cam respect to control cam ( mgus od : 97.1410 and mm od : 52.313.6 versus ctrl od : 158.715.52 ;
p<0.05 ) . immunoblotting analysis and quantification after western blot analysis of endostatin expression in cam treated with ec obtained from 3 mm and 3 mgus patients .
mm plasma cells secrete vegf , whereas both vegfr-1 and vegfr-2 are markedly elevated in the bone marrow stromal cells , suggesting that a paracrine growth pathway mediated by vegf - activated stromal cells , secretion of il-6 on their part , and subsequent activation of plasma cells may occur . in active mm , we found secretion of the vegf - a isoform by plasma cells , overexpression of vegfr-2 by bone marrow microvessels and isolated ec and of vegfr-1 by the other stromal cells , secretion of vegf - c and vegf - d by stromal cells , and expression of their cognate receptor vegfr-3 by plasma cells , which suggests another paracrine loop of angiogenesis and tumour growth in mm .
parallel studies by kumar et al . found secretion of vegf by several mm cell lines and fresh patients ' bone marrow plasma cells , as well as expression of vegfr-1 and vegfr-2 , which suggests multiple vegf - mediated autocrine pathways of tumour growth and paracrine stimulation of angiogenesis .
when we studied the angiogenic potential of plasma cell suspension obtained from patients with active mm and implanted them onto the cam , we demonstrated a vasoproliferative response , significantly higher than that induced by cell suspension obtained from patients either with non - active mm or mgus .
hgf / sf is yet another angiogenic factor identified in the culture medium of both human mm cell lines   and freshly isolated plasma cells .
still other factors may be responsible for bone marrow angiogenesis in mm : the plasma cell secretion products , such as tgf- , il-1 , il-6 and il-8 ; granulocyte - colony stimulating factor ( g - csf ) , granulocyte macrophage - colony stimulating factor ( gm - csf ) and tnf- , secreted by bone marrow microenvironment cells recruited and activated by plasma cells .
since human mm cell lines produce ang-1 that up - regulates the tie-2 cognate receptor in bone marrow ec , involvement of the ang-1/tie-2 loop in mm neovascularization has been demonstrated .
conversely , mmec secrete angiogenic cytokines , such as fgf-2 , vegf and ang-1 as well as matrix metalloproteinase-2 and -9 ( mmp-2 and mmp-9 ) and express adhesion molecules facilitating dissemination of these cells .
other studies , however , showed that the expression levels of vegf , fgf-2 , and their receptors were similar among plasma cells from mgus and mm , suggesting that increasing angiogenesis from mgus to mm is , at least in part , explained by increased tumour burden rather than increased expression of vegf / vegfr-2 by plasma cells .
another possibility is that the inhibition of angio - genesis in mgus is lost with progression , hence that the switch from mgus to mm may involve a loss of an anti - angiogenic activity .
our data are in accord with this statement , because the expression of endostatin mrna detected in both mgus and mm treated cam was significantly lower respect to control cam .
the phenotypic switch to angiogenesis in tumour growth involves more than simple up - regulation of angiogenic activity and is thought to be the result of a net balance of positive and negative regulators , and our study suggests that angiogenic switch in mm , investigated by using an in vivo experimental model such as the chick cam , may involve loss of an endoge - nous angiogenesis inhibitor , namely endostatin .

 abstractwe have attempted a fine characterization of the angiogenic response induced by multiple myeloma endothelial cells ( mmec ) by using the chick embryo chorioallantoic membrane ( cam ) assay and by reverse transcriptase - polymerase chain reaction ( rt - pcr ) .
 results showed that in the cam assay mmec induced an angiogenic response comparable to that of a well - known angiogenic cytokine , namely fibroblast growth factor-2 ( fgf-2 ) , while rt - pcr demonstrated that the expression of endostatin mrna detected in mm treated cam was significantly lower respect to control cam .
 these data suggest that angiogenic switch in mm may involve loss of an endogenous angiogenesis inhibitor , such as endostatin .


reverse shoulder prosthesis is an excellent surgical option for patients with certain shoulder pathologies , of which rotator cuff arthropathy is one .
one area where we are gaining a greater understanding is that of managing acromial pathology .
recent studies are showing that not all acromial pathology is a contraindication to reverse shoulder arthroplasty ; in both pre- and post - operative patients , small lateral fractures of the acromion can be treated non - operatively or fixed surgically with a tension band technique .
overall , results comparable to that of reverse arthroplasty can be achieved with either of these treatment options .
however , fractures at the base of the acromion or of the scapular spine present a different problem . in these types of fractures , a larger length of deltoid muscle is detached , and therefore defunctioned . in reverse shoulder arthroplasty , in order to achieve a stable shoulder , the deltoid must be tensioned a great deal .
this in turn causes a deforming force and distraction at the fracture site , making for a poor healing potential . also , as the reverse shoulder prosthesis relies on the deltoid muscle to move the arm , if the deltoid is detensioned or improperly tensioned , this will lead to poor functional capacity .
we present a case of treatment of a fracture at the base of the acromion using a 90/90 plating construct that healed in a good position .
a 71-year - old right - handed woman presented with a 2-year history of right shoulder pain and dysfunction , which began abruptly when lifting a heavy load . at the initial evaluation ,
her range of motion was limited , particularly in active abduction as she achieved only 45 degrees in this plane . a cuff arthropathy ( hamada grade 3 )
was diagnosed and the decision was taken to proceed with reverse shoulder arthroplasty . to complete the pre - operative assessment ,
a ct scan was performed which confirmed there was no acromial pathology involved . a reverse shoulder prosthesis and a biceps tenodesis
was carried out ( delta xtend , depuy , warsaw , in , usa ) through a deltopecoral approach [ figure 1 ] .
the patient 's right arm was immobilized in a sling and the range of motion exercises were started 6 weeks after arthroplasty [ figure 1a ] .
five months after surgery , she sustained a blow to the right shoulder but sought medical attention 1 month later , at her scheduled follow - up visit .
radiographs showed a displaced fracture at the base of the acromion [ figure 1 ] .
this showed that both the humeral and glenoid components were well fixed and the only pathology was the fracture .
antero - posterior radiographs showing the patient 's right shoulder immediately after reverse shoulder arthroplasty ( a ) and after fracture , 5 months later ( b ) .
the post - operative image shows no pre - existing acromial pathology she was taken to the operating room and her previous incision was extended into a  sabre  incision to better expose the acromion .
two small fragment ( 3.5 mm ) locking plates ( synthes , west chester , pa , usa ) were used in a 90/90 configuration ; one was a fragment specific clavicular plate and the other a reconstruction plate .
the fragment specific clavicular plate was applied on the superior edge of the scapular spine in a compression mode .
a second reconsrtuction plate was applied from the posterior acromion to the posterior cortex of the scapular spine , in the infraspinatus fossa .
fixation was solid , but in order to protect the construct , the patient was placed in an abduction brace for 6 weeks .
eighteen months after fracture fixation , the patient was satisfied with the clinical result [ figure 2 ] .
she was capable of 125 degrees of abduction , 160 degrees of forward flexion , 85 degrees of external rotation in adduction , and 60 degrees of internal rotation .
her quickdash score was 29.5 ( compared to 82.5 pre - op ) and constant score was 69 on the affected side compared to 85 on the left side , for a  good  functional outcome .
a medline search using the key words [ acromion fracture ] , [ scapula fracture ] , [ reverse arthroplasty ] was performed .
we then sought to classify and separate the cases by location ; there were 30 cases of fractures at the base of the acromion , and scapular spine . when our case is added , we have a total of 31 cases .
results are summarized in table 1 . in brief , of the 30 more proximal cases , 21 were treated non - operatively in a sling , 7 were treated with open reduction and open fixation and one with revision of the prosthesis . of those treated non - operatively , and for whom results are published , fourteen had a non - union , four had a malunion and in two cases , it was unclear whether union was achieved . for patients receiving fixation of the fracture , there was one non - union , two repeat fixations , and one patient required removal of the fixation .
the patient who had a revision of the prosthesis had a malunion of the acromion .
results of the literature review . in the fracture location column , the fracture description provided by the authors is presented .
although recent studies have shown that good outcomes may be achieved with non - surgical management of lateral acromial fractures , the same does not hold true for basal acromial fractures .
the likely reason for this is that with fractures at the base of the acromion , a large length of the deltoid is defunctioned and the deltoid muscle is essential to the functioning of the reverse shoulder prosthesis .
previously described techniques , such as tension band fixation achieved poor functional results for fractures at the base of the acromion .
we postulate that this is because the fixation method is insufficient to withstand the forces generated by the deltoid muscle  a tension band can neutralize forces parallel to the axis of the band but in the case of the deltoid , force vectors are generated in different directions .
it also allows for compression along the fracture site and resists motion in all directions .
good screw purchase can be achieved by angling the screws either toward the scapular spine or the coracoid .
the locking option enables good fixation and improves cut - out strength in osteopenic or osteoporotic bone .
in addition to the stable fixation achieved with this technique , we further recommend immobilization of the affected shoulder using a sling and an abduction pillow to detension the deltoid , thus at least partially removing the distractive forces at the fracture site . also essential to achieving a good functional result
we thus also recommend a program of range of motion exercises followed by strengthening exercises to maximize functional capabilities . based on results from previous studies ,
fractures of the tip were those of the most lateral or anterior portion of the acromion .
fractures of the body of the acromion are those medial to the tip of the acromion and lateral to the beginning of the scapular base .
the scapular base is the lateral border of the scapular spine , which is smooth and round . in our nomenclature ,
fractures at the scapular base are termed fractures at the base of the acromion as functionally , this zone connects the acromion to the rest of the scapula and this term avoids confusion with any more medially occurring fractures .
coronal ( a ) and axial ( b ) representations of the proposed classification scheme for acromial fractures associated with revere arthroplasty we propose the above mentioned classification system as it provides a nomenclature for acromial fractures that is descriptive and is based on the anatomy and functionality of the scapula . as the results from the review of the literature indicate , the more medial the fracture , the worse the prognosis with non - operative treatment .
thus , this classification system may be used to predict outcomes and determine treatment offered .
it differs from other classification systems ( crosby ) in that it further subclassifies more medial fractures , which are the more ominous fractures . as more research is carried out in this field

 fractures of the acromion and scapula are known to occur after reverse shoulder arthroplasty .
 we present a case of a fracture at the base of the acromion 5 months after arthroplasty treated successfully with dual plating of the acromion .
 eighteen months after fracture fixation , the patient had 160 degrees of active forward flexion , a quickdash of 29.5 , a constant score of 69 and she was satisfied with the result . a concomitant review of the literature produced , in addition to our patient , 56 cases .
 these were used to produce a classification system , based on bony and functional anatomy as follows .
 tip fractures are of the most lateral or anterior portion of the acromion , those of the body of the acromion are medial to the tip but lateral to the beginning of the scapular base
 . fractures at the scapular base are termed fractures of the base of the acromion and those more medial to that , fractures of the scapular spine .
 the functional results of these case series demonstrated poorer functional outcomes for more medial fractures .
 as future research in this domain increases , clarity on the nomenclature of these fractures will allow for prognostication and treatment based on fracture location as well as comparison between studies .


besides its classical role in calcium and bone homeostasis , vitamin d is considered a potent immunomodulator that can affect the pathogenesis of several autoimmune diseases .
our aim is to evaluate the effect of vitamin d correction to a patient with new onset graves disease ( gd ) with an underlying vitamin d deficiency .
we describe the effect of vitamin d3 on untreated graves disease with vitamin d deficiency .
a healthy saudi woman in her 40s sought consultation with a three - month history of palpitation .
she denied any history of heat intolerance , weight loss , menstrual irregularity or sweating .
. physical examination revealed a mild diffusely enlarged and non - tender thyroid gland with no bruit .
the initial thyroid function test , which was done in an outside hospital , revealed a tsh , 0.01 miu / l ; ft4 , 22.5
vitamin d 25-oh level was done in our hospital and showed a result of 26.0 nmol / l with a tsh , 0.013 miu / l ; ft4 , 16.7
tc-99 m thyroid scintigraphy demonstrated an enlarged thyroid gland with increased radiotracer trapping and heterogeneous distribution .
the patient was given only oral cholecalciferol 4000 iu per day since november 2012 ( prescribed by an outside hospital ) then from may 2013 onwards she was given 50,000 iu per month .
follow - up laboratory exams revealed improved vitamin d levels as well as tsh and ft4 .
vitamin d deficiency may exacerbate the onset and/or development of gd and correction of the deficiency may be able to reverse it .
however , further prospective clinical studies will be needed to define the role of vitamin d treatment in gd .
more recently , vitamin d has been shown to be a modulator in both innate and adaptive immunity.1 there is a well - established link between vitamin d deficiency and various autoimmune diseases , including type 1 diabetes mellitus ( t1 dm ) , systemic lupus erythematosus ( sle ) , rheumatoid arthritis ( ra ) , inflammatory bowel disease ( ibd ) , and multiple sclerosis ( ms ) .
furthermore , it has been found that the supplementation of vitamin d can prevent the onset and/or development of different kinds of autoimmune disorders in human beings and animal models.2 in addition , it has been shown that the prevalence of vitamin d deficiency is common in patients with graves disease ( gd),3 and is associated with higher thyroid volume.4 in our case report , we evaluated the effect of vitamin d correction to a patient with new onset gd with an underlying vitamin d deficiency .
a healthy saudi woman in her 40s sought consultation with a 3 months history of palpitation .
she denied any history of heat intolerance , weight loss , menstrual irregularity , diarrhea , or sweating .
there was no personal or family history of thyroid disease and no specific medication history .
physical examination revealed a mild diffusely enlarged and non - tender thyroid gland with no bruit .
the initial thyroid function test , which was done in an outside hospital , revealed a tsh , 0.01 miu / l ; ft4 , 22.5
vitamin d 25-oh level was done in our hospital and showed a result of 26.0 nmol / l with a tsh , 0.013 miu / l ; ft4 , 16.7
anti - thyroid antibodies showed a tg , 17.1 iu / ml ; tpo , 0.19 iu / ml with a positive tsh receptor antibody .
tc-99 m thyroid scintigraphy demonstrated an enlarged thyroid gland with increased radiotracer trapping and heterogeneous distribution ( fig .
the patient was given only oral cholecalciferol 4000 iu per day since november 2012 ( took it from an outside hospital ) then from may 2013 onwards she was given 50,000 iu per month .
the serial thyroid function tests , vitamin d levels , and titer autoantibodies are summarized in table 1 .
follow - up laboratory exams revealed improved vitamin d levels as well as tsh and ft4 .
written informed consent was obtained from the patient for the publication of this case and accompanying images .
it has become apparent that multiple factors contribute to the etiology of gd , including genetic and environmental factors .
these activated t cells in turn increase the secretion of thyroid - specific autoantibodies from b cells .
the prevalence of vitamin d deficiency was reported to be common in patients with gd.3 whether vitamin d deficiency has a causal relationship with gd remains a controversial issue .
misharin et al.5 observed that vitamin d deficiency was found to modulate graves hyperthyroidism induced in balb / c mice by thyrotropin receptor immunization . in this study ,
balb / c mice on a vitamin d deficient diet were more likely to develop persistent hyperthyroidism than other mice receiving adequate vitamin d supply . in another study ,
combination treatment with methimazole and vitamin d3 ( 1,25 ( oh)2d ) in patients with gd has more rapid euthyroidism achievement compared with patients receiving methimazole alone.6 in addition , vitamin d supplementation has been shown to inhibit inflammatory responses in human thyroid and t cells.7 interestingly , vitamin d deficiency is found to be associated with higher thyroid volume in patients with newly onset gd.4 it has been recently discovered that vitamin d - receptor gene and vitamin d - binding protein gene polymorphisms are associated with gd.8,9
our present case supports the current literature and strongly suggests that vitamin d deficiency may exacerbate the onset and/or development of gd and correction of which may be able to reverse it .
however , further prospective clinical studies will be needed to define the role of vitamin d treatment in gd .

 objectivebesides its classical role in calcium and bone homeostasis , vitamin d is considered a potent immunomodulator that can affect the pathogenesis of several autoimmune diseases .
 our aim is to evaluate the effect of vitamin d correction to a patient with new onset graves disease ( gd ) with an underlying vitamin d deficiency.methodwe describe the effect of vitamin d3 on untreated graves disease with vitamin d deficiency.resultsa healthy saudi woman in her 40s sought consultation with a three - month history of palpitation .
 she denied any history of heat intolerance , weight loss , menstrual irregularity or sweating .
 she has a history of chronic muscle aches and pains . physical examination revealed a mild diffusely enlarged and non - tender thyroid gland with no bruit .
 she had no signs of graves ophthalmopathy . in laboratory examinations ,
 the initial thyroid function test , which was done in an outside hospital , revealed a tsh , 0.01 miu / l ; ft4 , 22.5
 pmol / l and ft3 , 6.5 pmol / l .
 vitamin d 25-oh level was done in our hospital and showed a result of 26.0 nmol / l with a tsh , 0.013 miu / l ; ft4 , 16.7
 pmol / l ; and ft3 , 3.8 pmol / l .
 tsh receptor antibody was positive .
 tc-99 m thyroid scintigraphy demonstrated an enlarged thyroid gland with increased radiotracer trapping and heterogeneous distribution .
 the patient was given only oral cholecalciferol 4000 iu per day since november 2012 ( prescribed by an outside hospital ) then from may 2013 onwards she was given 50,000 iu per month .
 follow - up laboratory exams revealed improved vitamin d levels as well as tsh and ft4 .
 she eventually improved both clinically and biochemically with a satisfactory outcome.conclusionvitamin d deficiency may exacerbate the onset and/or development of gd and correction of the deficiency may be able to reverse it .
 however , further prospective clinical studies will be needed to define the role of vitamin d treatment in gd .


 nigella sativa l. ( ns ) is a vegetal specie of the ranunculaceae family , commonly known as  black cumin seed  ,  neguilla  or  ajemuz  , that is   widely cultivated in the mediterranean region .
its seeds have played an important role over the years in ancient islamic system of herbal medicine and in spain , where they have been traditionally used in folk medicine .
ns seeds have shown several therapeutic effects such as prevention of cancer , antihypertensive effect , anti - inflammatory , analgesic , and antihistaminic   actions .
the volatile oil from this plant presents a relaxant action on different smooth muscles   and tracheal muscles of guinea pigs .
there is evidence of anticonvulsant and antioxidant effects against pentylenetetrazol - induced kindling in mice .
. likewise , aqueous extract of this plant suppresses penicillin - induced epileptic activity in rats .
this anticonvulsant effect is a consequence of selectively altering the monoamine level in different brain regions . in recent works , histopathological changes of neurodegeneration in the frontal cortex and brain stem in neurons after exposition to toluene have been observed [ 15 , 16 ] .
the administration of ns extract and thymoquinone ( major component from ns volatile oil ) causes morphologic improvement over apoptosis and indicates that ns therapy is useful as a potential treatment of neurodegeneration prevention .
ns seeds composition includes nutritional components such as carbohydrates ( glucose , xylose , rhamnose , and arabinose ) , vitamins as thiamine , riboflavin , pyridoxine , niacin and folic acid , mineral elements , and proteins .
the ns seeds are also a source of calcium , iron and potassium , alkaloids ( nigellidine , nigellimine , and nigellicine ) , 36%38% fixed oil and 0.4%2.5% essential oil . the fixed oil is mainly composed of unsaturated and essential fatty acids ( linoleic acid , followed by oleic acid )   whereas the volatile oil has   been shown to contain 18.424% thymoquinone and 46% monoterpenes such as p - cymene and pinene [ 5 , 20 ] .
thimoquinone , as indicated above , is thought to be the main active component of ns seeds    and suppresses itself epileptic seizures in rats , while a monodesmosidic triterpene saponin , -hederin , has also been isolated from the extract of ns seeds and proved to exert antitumoral activity .
three flavonoid glycosides and triterpene saponins were also identified from nigella sativa , together with four phospholipid classes : phosphatidylcholine   phosphatidylethanolamine , phosphatidylserine , and phosphatitdylinositol [ 24 , 25 ] . in previous studies we demonstrated that aqueous and methanolic extracts of ns seeds exert a potent sedative and depressive effect on cns and induce analgesia .
the effect on cns resulted in a significant reduction of spontaneous motility , a decrease in normal body temperature and significant analgesic action against hot - plate and pressure tests .
body temperature reduction can be interpreted as an index of alteration of various central neurotransmitters ; anxiety and sedation are mainly mediated by the gaba - a receptor in the cns . since depressant action was confirmed specially for the methanolic extract , we decided to study whether the addition of this methanolic dry extract in cortical neurons culture could exert any influence on the secretion of the excitatory amino acids aspartate ( asp ) and glutamate ( glu ) , and the inhibitory amino acids gaba and glycine ( gly ) , as well as the presence of these amino acid neurotransmitters in the extract .
we also considered its effect on the amino acids secretion when stimulated by a depolarizing agent and its effect on cultured neurons viability .
the viability of the cultured neurons after exposition to ns extract concentrations 2.5 , 25 , and 250  g / ml during 15 and 60  minutes are shown in figure 1 .
results are expressed as a percentage with respect to control value ( 100% viability ) .
the amino acids gaba , gly , glu , and asp were measured in ns methanolic extract by hplc .
the results are expressed as pmoles / mg of ns extract ( table 1 ) .
the results showed gly as the most abundant , followed by gaba , glu , and asp , respectively .
the amino acids content in the extract was subtracted from the total amino acid content measured in the cellular medium after neuronal stimulation with ns extract so we can state that the final amino acids content in cell culture is a direct consequence of cell release .
the release of the four amino acids after stimulation with the chosen nontoxic concentrations of ns extract ( 2.5 , 25 , and 250  g / ml ) showed a diminished secretion response that was statistically significant ( p < .05 for gly and glu ; p < .01 for gaba ; p < .001 for asp ) after 15  minutes of incubation with respect to control ( neuronal cells stimulated with normal locke medium during the same period of time and considered as 100% secretion ) ( figures 2 , 3 , 4 , and 5 ) .
gly and asp release was reduced in a dose   dependent manner ; glu and gaba showed a tendency to retrieve control values , although their secretion was lower than control .
this fall of aminoacids release is greater for higher extract concentration except for glu and gaba , which showed a tendency to recuperation to control values at the same time    ( figure 2 ) .
the hplc analysis revealed the same behavior for all the amino acids , with the exception of gaba , after treatment with ns extract during 60  minutes .
the increased presence of this amino acid was statistically significant for 25 and 250  g / ml ns extract . in order to know the response to a depolarizing agent ,
cortical neurons were stimulated with ns extract at the indicated concentrations , during 15 and 60  minutes previous to depolarization with 60 mm kcl ( figures 6 and 7 ) .
the neurons treated with ns extract during 15  minutes and subsequently stimulated with kcl showed a dose - dependent decrease in amino acids secretion with respect to control value ( neuronal cells stimulated with locke medium ) , which was considered as 100% . the observed behaviour was more relevant for glu and asp at 25 and 250   g / ml than for gaba and gly under the same conditions ( figure 6 ) .
measurement of secretion mediated by kcl during 60  minutes revealed an inhibition of the liberation of these neurotransmitters .
in this case , only gaba and glu were released in a dose - dependent manner ( figure 7 ) .
the aim of the study was to determine the effects of ns methanolic extract on the release of neurotransmitter amino acids by measuring their concentrations in the culture media using hplc precolumn derivatization technique .
three concentrations of ns extract ( 2.5 , 25 , and 250  g / ml ) and two time points ( 15 and 60  min ) for the determination of the effects were used .
this is a preliminary study which shows that exposure of the cultured neurons have a modulatory effect on the release and contents of these aminoacids .
the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide ( mtt ) assay was employed to estimate the cells viability when neurons were treated with ns extract .
the three concentrations of dry methanolic extract used in our study did not affect cellular respiratory capacity at any of the two periods of time considered .
these results allowed us to consider the adequate approach to the study of amino acid secretion in the conditions selected and to confirm the innocuous characteristics of the chosen extract concentrations .
our previous in vitro findings support the hypothesis that the sedative and depressive effects of nigella sativa ( ns ) observed in vivo , could be based on changes of inhibitory / excitatory amino acids levels .
several authors attribute the sedative effects of different plant extracts ( valeriana officinalis l. , scutellaria lateriflora l. ) [ 27 , 28 ] to its endogenous gaba concentration , although they also hypothesize that there exist other components of the vegetal extracts with benzodiazepine - like effects that may account for their in vivo effects .
the aim of our research was to determine whether these substances that are present in the ns extract could mediate the specific effects previously observed in vivo through the secretion of asp , glu , gaba , and gly .
first of all , the presence and content of the four amino acids in the extract was analyzed by hplc , showing a major presence of inhibitory amino acids ( gly and gaba ) ( table 1 ) .
then , the cell amino acids release was assayed after addition of several concentrations of ns extract to cultured neurons during two different periods of time ( 15 and 60  minutes ) .
the aim of this approach was to observe whether neuronal secretion could be modified depending on the exposition time .
high secretion of gaba was observed after 60  minutes ' contact with ns extract at 25 and 250  g / ml . this time is close to the one that allowed the maximum effect in our in vivo study , close to 40  minutes . under these conditions ,
similar results have been observed when high gaba concentrations are present in an aqueous extract of valeriana officinalis root which induced in vitro liberation of [ h]gaba in rat synaptosomes by reversal of the gaba carrier and inhibition of its reuptake .
both plants are widely prescribed as sedative / anxiolytic ones . as the gaba presence in the methanolic extract of ns
was confirmed with our results , it is possible to assume that a high level of gaba in the medium during cellular incubation and a longer period of exposition at this one could exert the same effect above described .
recent findings suggest that ns protects from induced generalized epilepsy in rats by selectively altering the monoamine level in different brain regions .
this study pointed that ns possibly facilitates the inhibitory activity of the gabaergic system through a competitive agonist action in the benzodiazepine ( bzd ) site of the gaba receptor as well as the involvement of dopaminergic and noradrenergic system .
the confirmed presence of gaba in the methanolic extract could be directly related to its conduct over the gaba receptor and this could explain the potent sedative and depressive effect on cns as previously reported . moreover , the important presence of gly in the extract could be also related to its inhibitory action .
the binding of both neurotransmitters to their receptors on the neuronal membrane induces hyperpolarization that could be responsible for a significantly lower secretion of amino acids with respect to control values .
in addition to this , our results showed a decrease in neuronal excitatory activity derived from a diminished asp and glu secretion , specially the second one .
our results show the behavior of the cortical neurons and confirm what we expected from the previous study performed in vivo .
likewise the methanolic extract composition is able to mediate in the neuronal amino acids release . in this case , the ns extract had an influence on neuronal transmission because it modifies the neurotransmitter amino acids release .
depolarization of neuronal cells by application of high k+ concentration ( 60  mm kcl ) induced a diminished amino acid release in both periods of time assayed .
this diminished release was similar to nondepolarizing secretion except for gaba which also diminished its secretion .
although multiple mechanisms of neurotransmitter release evoked by elevated extracellular k+ may be involved [ 29 , 30 ] , we consider that the effect of ns extract over neuronal cells produces a drop in the transmission and is responsible for of the inhibitory effect indicated above .
a possible action of ns extract over l - type calcium channels or an opening on potassium channels had been suggested by others authors [ 3135 ] , and it could contribute to the relaxant activities of this plant .
furthermore , its possible effect over these channels could persist even after the extract was eliminated from the medium before depolarization .
with respect to gaba secretion , the only amino acid which differs in its behavior with respect to nondepolarizing liberation , the diminished secretion may derivate from the membrane transporter - reversal for gaba above suggested . in our study , we have considered the possibility that the gaba carrier had been previously affected by the presence of ns extract during 15 or 60  minutes , inhibiting gaba reuptake and favouring its liberation from neurons .
other authors   have concluded that extracellular k+ ( 50  mm kcl ) provokes gaba release by reversal transporter of gaba . in our case
, we observed a diminished liberation that could be justified by the previous loss of cellular gaba content as the neurons had been treated with the ns extract . in conclusion , this study suggests a sedative effect of ns methanolic extract by modification of neurotransmitter amino acids release , because the ns extract may induce an important release of gaba and gly in the cultured neurons medium and therefore , exert an increase in the agonist action over their receptors .
the results explain the sedative and depressive effects observed in vivo by an increase in inhibitory amino acids at the synaptic terminals .
even more , this effect is complemented with a possible decrease of excitatory transmission , as it has been demonstrated in vitro and could contribute to inhibitory response .
minimum essential eagle 's medium ( emem ) was obtained from bio - whittaker , and foetal bovine serum ( fbs ) and horse serum ( hs ) were procured from sera - lab ( sussex , england )
. standards of glutamate , aspartate , gaba and glycine were purchased from sigma ( st . louis , mo , usa ) .
reagents and solvents for hplc were triethylamine from sigma ( st . louis , mo , usa ) , acetic acid and methanol ultra gradient grade from merck ( darmstadt , germany ) .
distilled water used for the preparation of buffers and standards was deionized with milli - q purification system .
syringe filters millex - gv were obtained from millipore ( milford , ma , usa ) .
membrane filters ( 0.45  m pore size ) from tecknochroma ( barcelona , spain ) were used for filtration of the mobile phase and samples .
cell proliferation kit ii ( xtt ) , colorimetric determination , was purchased from roche diagnostics gmbh ( mannheim , germany ) .
, usa ) coupled with a photodiode array detector shimadzu spd-10a ( izasa , madrid , spain ) was used for the amino acids isolation and quantification . the analytical system consisted of a waters ods spherisorb 150  4.6  mm i.d . ; 5  m packed column ( teknokroma , barcelona , spain ) as stationary phase , preceded by a guard column spherisorb rp-18 , 5  m , 4  mm  4 mm .
a microplate fluorescence reader fl600-biotek spectrofluorimeter was used for the quantification of cell viability at 492  nm .
seeds of nigella sativa l. were supplied by the medicinal and aromatic plants research institute of egypt ( el cairo , egypt ) .
herbarium samples were authenticated by a taxonomist and a voucher specimen was deposited in the herbarium of the faculty of pharmacy , universidad complutense de madrid , with voucher number maf 161043 .
the methanolic extract of the plant was prepared according to ( science and technology program for development ) cyted protocol for vegetal species from countries that are included in this program .
brain neurons were obtained from foetal rat brains of 19 days of gestation as previously described   with minor modifications .
isolated neurons were suspended in eagle 's minimum essential medium ( emem ) containing 0.3  g / l glutamine , 0.6% glucose , 5% phosphate buffered saline ( pbs ) , 5% horse serum ( hs ) , 100  u / ml penicillin and 100  g / ml streptomycin .
cells , at a density of 1  10 cells / well , were placed on plastic petri dishes of 24 wells , treated with 10  g / ml poly - l - lysine to aid attachment .
the plates were incubated in a humidified incubator in an atmosphere of 5% co2/95% air at 37c . after 72  hours ,
nonneuronal cells ( contaminating glial cells ) were mitotically inhibited by exposure to cytosine arabinoside .
the incubation medium was replaced by fresh medium to which cytosine arabinoside was added to a final concentration of 10  m .
after 3 days , this medium was replaced by fresh medium and experiments were carried out using cultures ranging from 1015 days .
cell purity was checked by both cells staining with cresyl violet to identify neurons and with the specific antiglial fibrillary acidic protein ( gfap ) antibody to identify glial cells .
cortical neurons , after 7 days in culture , were detached from the culture plates with trypsin solution ( 0.25% trypsin and 0.02% edta in dulbecco 's buffered saline without calcium and magnesium ) and then the cells were fixed ( during 30  min ) with 2% p - formaldehyde .
after two washes with 1  ml of pbs , the cells were treated ( during 1  h ) with anti - gfap antirabbit ( at dilution of 1/500 ) .
subsequently , the cells were washed with pbs and treated with antirabbit conjugated igg fitc , at 1/100 , for 30  min .
the glial cells in the cultures were 8.3  3.6% of the total ( neural + glial cells ) .
this assay is used as an index of cell survival or cellular respiratory capacity - based on method of mossmann   and improved by weislow et al .   and roehm et al . .
the tetrazolium assay is based on the mitochondrial dehydrogenases ( md ) activity and their inactivation after cell death . in live cells ,
mtt is reduced to a highly water - soluble orange colored product , formazan dye .
neuronal cells were seeded in 96-multiwell plates at a density of 36  10 cells / well ( in 200  l medium ) and kept in the incubator until 8090% confluence .
after this , the medium was removed and the cells were washed twice with pbs and the ns extract previously dissolved in pbs was added to the wells and incubated for 15  minutes or 1  hour .
after each one of the treatment periods ( 15 or 60  min ) , the medium was removed and incubated with mtt solution ( final concentration 0.3  mg / ml ) , according to the kit specifications .
after 2  h incubation at 37c in a humidified atmosphere , orange dye solution was spectrophotometrically quantified using an elisa plate reader at 492  nm .
the amount of orange formazan formed , as monitored by the absorbance , directly correlates to the number of living cells .
hplc analysis of amino acids was performed by a previously described method   with minor modifications and with the equipment and conditions that were previously developed for the amino acids determination .
prior to hplc amino acid secretion analysis cells were washed twice , at 10  min intervals , with 1  ml of locke medium . after removing the medium ,
cells were stimulated for 15  min or 1  hour at 37c with 250  l fresh locke medium ( control cells ) or with 250  l of locke medium containing the dry methanolic extract of ns seeds at different concentrations ( 2.5 , 25 and 250  g / ml ) .
after stimulation , the wells medium ( supernatant s1 ) was taken for amino acid valuation and cells were then stimulated with 250  l of 60  mm kcl for 15  min .
after this , the cells secretion ( supernatant s2 ) was removed for their valuation and cells were lysated with 250  l of distilled water for total intracellular amino acids content determination .
the different supernatants obtained , s1 and s2 , as well as the cell lysated , were lyophilized for their dansylation .
the content of the four amino acids present in the methanolic extract was also determined by hplc analysis under the same conditions and these results were subtracted from the total amino acids content in cellular medium in presence of ns extract ( s1 ) in order to obtain cellular secretion value itself .
the results were calculated as the amino acid release into the incubation medium with respect to the total amino acid content .
these results were expressed as a percentage of secretion with respect to control experiments that were considered as 100% .
data are presented as means  sem of four separated experiments from different cell cultures , each one performed in triplicate with different batches of neuronal cells .

 nigella sativa l. ( ns ) has been used for medicinal purposes since ancient times . this
 study aimed to investigate the cytotoxicity of ns dry methanolic extract on cultured
 cortical neurons and its influence on neurotransmitter release , as well as the presence of
 excitatory ( glutamate and aspartate ) and inhibitory amino acids ( gamma - aminobutyric
 acid  gaba  and glycine ) in ns extract .
 cultured rat cortical neurons were exposed to
 different times and concentrations of ns dry methanolic extract and cell viability was
 then determined by a quantitative colorimetric method .
 ns did not induce any toxicity .

 the secretion of different amino acids was studied in primary cultured cortical neurons
 by high - performance liquid chromatography ( hplc ) using a derivation before injection
 with dansyl chloride .
 ns modulated amino acid release in cultured neurons ; gaba was
 significantly increased whereas secretion of glutamate , aspartate , and glycine were
 decreased .
 the in vitro findings support the hypothesis that the sedative and depressive
 effects of ns observed in vivo could be based on changes of inhibitory / excitatory amino
 acids levels .