[Analytical performance verification protocols and performance specifications of platelet-dependent von Willebrand factor activity testing].

Objective: To investigate the analytical performance verification protocols and performance specifications of platelet-dependent von Willebrand factor (VWF) activity testing (VWF:GPIbM) for clinical laboratories. Methods: According to Clinical Laboratory Standards Institute (CLSI) documents and National Health Standard of China, the performance verification of VWF:GPIbM was designed and implemented using Sysmex CS-5100 instrument and its corresponding reagents. (1) Precision verification: Two commercial quality control samples (with normal and low activity levels) and three plasma pools (with activity range from 5.0% to 150.0%) were prepared. Each sample was tested five times daily for five consecutive days. The coefficient of variation (CV) of intra-and inter-run precisions were calculated, and the precision evaluation criterion was set according to package inserts. (2) Trueness verification: The calibrator was diluted to five reference materials with activity values of 5.2%, 31.2%, 62.4%, 104.0% and 138.7%, and each reference material was tested five times daily for five consecutive days. The bias between the measured value and the reference value was calculated, and the trueness evaluation criterion was set according to the total allowable error. (3) Linearity verification: Ten pooled plasmas with theoretical value range from 3.6% to 160.4% were prepared for the linearity verification of two calibration curves set by the manufacturer (i.e. low range and normal range calibration curve). Each pooled plasma was tested three times in a single run. The slope and R2 of linear regression of mean of measured value and theoretical value, as well as bias, were calculated, and the linearity evaluation criterion was set according to National Health Standard of China and package inserts. (4) Limit of quantitation verification: The calibrator was diluted to two reference materials with activity values of 3.3% and 2.7%, and each material was tested twelve times. The limit of quantitation evaluation criterion was set according to CLSI document. Results: The CVs of intra-and inter-run were 1.0%-2.5% and 1.1%-2.6%, respectively. The biases of trueness verification were -0.4%, 1.0%, -2.6%, 0.3% and -2.7%, respectively. The linearity verification results of low range (3.6%-31.8%) and normal range (28.4%-160.4%) showed that the slopes of regression equation were 1.021 7 and 0.996 2, respectively, R2 were 0.993 5 and 0.993 9, respectively, and the biases with 0-1.8% and -10.1%-0 of plasmas met the criterion. The biases ranged from -0.4% to 0.3% of test results in the verification of limit of quantitation met the criterion. Conclusion: The verification results of VWF:GPIbM assay for precision, trueness, linearity and limit of quantification meet the performance requirements indicated in the package inserts and the criteria set in this study, which can be taken as a reference of performance verification for the clinical laboratories.

[Current status of CD34+cell enumeration assay in clinical laboratories and its improvement].

Objective: To investigate the current status and problems of CD34+ cell enumeration in clinical laboratories and provide suggestions for the development of quality improvement programs. Methods: A total of 101 laboratories participating in the national external quality assessment program of CD34+cell enumeration were surveyed. Questionnaires and quality assessment materials were distributed to collect information on assay methodology and testing results. Quality control requirements for CD34+cell enumeration were determined according to the international guidelines, and the compliance of the surveyed laboratories was analyzed. Testing results were analyzed in groups and compared with the College of American Pathologists (CAP) quality assessment data. Results: A total of 97 laboratories returned the questionnaires and 99 laboratories returned the results of quality assessment materials. The questionnaire data showed high compliance rates of quality control requirements such as gating protocols, pipetting methods, and the number of cells acquired (92.8%, 83.9%, and 82.5% respectively). However, these laboratories had relatively low compliance rates such as the use of whole blood quality control materials for internal quality control, selection of erythrocyte lysing reagents, sample processing method, whether to report absolute count results, and quality control of equipment (5.2 %, 28.9%, 39.2%, 46.4%, and 55.7%, respectively). Testing results demonstrated that the coefficient of variation (CV) of percent counts was similar to the CAP quality assessment data, but the CV of absolute counts was greater than the CAP quality assessment data. Conclusions: Clinical laboratories have poor compliance with some quality control requirements and the variability of absolute count results between different laboratories is not satisfactory. Therefore, it is recommended that clinical laboratories should strengthen the training related to the quality control of CD34+cell enumeration, especially the absolute counting.

[Clinical application of the simultaneous detection of methotrexate and 7-hydroxymethotrexate in the delayed elimination for pediatric acute lymphoblastic leukemia].

Objective: To discuss the application value of the simultaneous determination of methotrexate (MTX) and 7-hydroxymethotrexate (7-OHMTX) in the delayed elimination of MTX for pediatric acute lymphoblastic leukemia (ALL). Methods: Cross sectional study. A total of 97 children who received 192 high-dose MTX treatments cycles in Lu Daopei Hospital from April to August 2019 were enrolled. The peripheral blood was collected at 0,24,48 h after the end of MTX infusion and analyzed by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). One hundred and ninety-two MTX treatments were divided into a normal MTX elimination group (n=149) and delayed elimination group (n=43) according to the standard of delayed elimination and divided into 0-9 year old group (n=95), 10-14 group (n=50), 15-18 group (n=47) according to age. The comparisons of the C(MTX), C(7-OHMTX) between normal and delayed group was conducted as well as among different age groups. Receiver operator characteristic curve (ROC) of C(MTX-0h) and C(7-OHMTX-0h) was analyzed and the concentration corresponding to the maximum of the Youden index on the ROC was set as the warning value for delayed elimination. Correlation between the delayed elimination after the end of MTX infusion and toxicity was investigated and the percentage of delayed elimination was also analyzed. Results: The concentrations of MTX and 7-OHMTX were significantly higher in the delayed elimination group than the normal group. Immediately after infusion (0 h), a C(7-OHMTX-0h) of >17.8 µmol/L (sensitivity 97.7%, specificity 54.4%) and a C(MTX-0h) of >148.8 µmol/L (sensitivity 72.1%, specificity 84.6%) were found to be warning predictors of delayed elimination under the MTX treatment protocol. MTX delayed elimination was positively correlated with methotrexate-induced toxicities (r=0.58, P<0.01). The percentage of hepatotoxicity and nephrotoxicity was 32.6% and 37.2% in the delayed elimination group, which was significantly higher than normal group of 12.8% and 3.4% (P<0.05). No significant difference was found in other toxicities. There was significant difference in C(MTX) among different age groups but no significant difference in C(7-OHMTX). Conclusion: Simultaneously determination of MTX and 7-OHMTX in plasma by HPLC-MS/MS in childhood ALL patients can provide a reference for clinical individualized medicine and pharmacokinetic research.

[Association study of genetic variations in SLCO1B3 gene with prognosis in breast cancer patients treated with neoadjuvant chemotherapy of TA regimen].

Objective: To assess the association of single nucleotide polymorphisms (SNPs) in SLCO1B3 gene with prognosis of breast cancer (BC) patients treated with neoadjuvant chemotherapy of TA regimen (taxane and antharcycline drugs). Methods: 439 female BC patients were recruited and treated with neoadjuvant chemotherapy of TA regimen. A blood sample (2 ml) of peripheral blood was collected from each patient before chemotherapy. Tagging SNPs (tag-SNPs) were selected. We investigated the association of tag-SNPs with prognosis, by Sequenom Mass ARRAY system platform, characterizing tag-SNPs. The hazard ratio (HR) and 95% confidence interval (CI) for progression or death were calculated by multivariable-adjusted Cox regression model. Results: Seven tag-SNPs (rs11045689, rs200104106, rs3764006, rs3834935, rs4149117, rs7305323 and rs73241801) were selected for study. Compared with individuals carrying the rs11045689 GG genotype, individuals carrying rs11045689 AA genotype performed worse PFS and OS, with the HR (95% CI) for progression being 1.39 (1.11~1.75) and the HR (95% CI) for death being 1.38 (1.04~1.83). Compared with individuals carrying the rs73241801 CC genotype, individuals carrying rs73241801 TT genotype performed better OS (P=0.041), with the HR (95% CI) for death being 0.65 (0.44~0.94). The number of risk allele was significantly associated with PFS (P=0.012) and OS (P=0.017) of BC patients by accumulation analysis. Compared with individuals carrying one or less than one risk allele, individuals carrying four risk alleles performed worse PFS and OS, with the HR (95% CI) for progression being 1.37 (1.09~1.72) and the HR (95% CI) for death being 1.36 (1.02~1.81). Conclusion: The variations of rs11045689 and rs73241801 in SLCO1B3 gene were significantly associated with prognosis of BC patients treated with neoadjuvant chemotherapy of TA regimen, which might serve as biomarkers for predicting prognosis of BC patients treated with neoadjuvant chemotherapy.

[Study on commutability evaluation of reference materials for fibrinogen].

Objective: To discuss the commutability evaluation method of reference materials for fibrinogen measurement and evaluate the commutability of the Third WHO International Standard Fibrinogen Plasma (WHO 09/264), SSC/ISTH Secondary Coagulation Standard (SSC LOT4) and homemade reference materials (RM01, RM02) in order to provide suggestions on how to determine the suitable method of commutability evaluation and reliable traceability standard. Methods: The comparability of fibrinogen among different measurement systems were evaluated and WHO 09/264 was used to calibrate each system to improve the comparability if the comparability among different systems couldn't be accepted. Forty clinical samples and the reference materials randomly interspersed among the clinical samples were measured on Stago STA-R Evolution, Sysmex CS 5100, IL ACL TOP 700 simultaneously. Measurement results were pairwise analyzed by Deming regression and difference in bias approach according to the Clinical and Laboratory Standards Institute (CLSI) EP14-A3 protocol and the recommendations of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) working group on commutability, respectively. Results: The comparability of fibrinogen measurement among common systems could not meet the criterion. WHO 09/264 could improve the agreement among different measurement systems. The prediction interval of Deming regression was affected by the comparability of measurement systems, resulting in unreliable results. The difference in bias approach was more suitable because its criterion was related to the medical requirements. WHO 09/264 was commutable between Stago and Sysmex, inconclusive between Stago and IL, Sysmex and IL (The calibration effectiveness of WHO 09/264 showed that it was commutable among the three measurement systems). SSC LOT4 was commutable between Stago and Sysmex, inconclusive between Stago and IL, Sysmex and IL. RM01 and RM02 were commutable between all systems pairs assessed by difference in bias approach. Conclusions: There are differences in the results of two commutability evaluation approaches. The difference in bias approach is recommended for commutability evaluation. WHO International Standard and homemade reference materials can be used as traceability standard for fibrinogen measurement.

[Analysis of internal quality control data of complete blood count from laboratories participating in national external quality assessment].

Objective: To investigate current status and problems of internal quality control (IQC) of complete blood count in China so as to perform IQC normally. Methods: The IQC data of complete blood count for five parameters were collected from laboratories participating in national external quality assessment during 2012-2017 (totally 12 times), including WBC, RBC, Hb, Hct and PLT. After confirmation of all data, data for the 12 times were analyzed as follows.The proportions of using different levels of quality control materials were calculated.The 25th, 50th, 75th, 90th percentiles CV of data collected for the 12 times were calculated respectively and the trends of CV were observed over time.The difference of CV among laboratories running three control levels was compared.The CV of each parameter in 2017 was compared with precision requirements based on biological variation, health standards and German Medical Association Directive; The proportions of laboratories using different control rules were calculated. Results: After invalid data was excluded from those IQC data of laboratories for the 12 times external quality assessment (up to 2 402, as low as 1 449) from 2012 to 2017, the residual data (up to 2 332, as low as 1 431, accounting for 96.0%-99.2%) was used for analysis. 61.9%-66.1%, 18.2%-23.6% and 14.3%-17.3% of laboratories ran one, two and three control levels respectively, and the proportions of laboratories running more than two control levels increased from 33.9% to 38.1%. The decrease trend of the 75th, 90th percentiles CV of WBC, RBC, Hb, Hct for three levels, PLT for normal level and the 90th percentiles CV of PLT low level had statistically significance over time (P<0.05); the decrease trend of the 75th percentiles CV of PLT low level and 75th, 90th percentiles CV of PLT high level had no statistically significance over time. The CV had significant difference between low and normal, low and high control level for WBC and PLT, while there were no difference between normal and high control levels. There were no significant difference of CV among three control levels for RBC, Hb, and Hct. Except for the CV of Hct low, normal level and PLT low level, 85% of laboratories for the other parameters could meet the minimum precision requirements based on biological variation; more than 85% laboratories met the requirements of health standards; except for the CV of PLT low level, more than 80% laboratories met the requirements of German Medical Association Directive. The proportion of laboratories using 1(3s)/2(2s) quality control rules increased from 59.2% to 76.0%. Conclusions: During the past 6 years, the CV for IQC has shown a decrease trend over time. However, the control level and quality control rules used by some laboratories do not meet management requirements. The CV of Hct and PLT in a few laboratories do not meet the minimum requirements of the health standards, and need to implement quality improvements fatherly.

[Study and analysis of national current status and problems of anticoagulant proteins assay].

Objective: To investigate current status and problems of anticoagulant proteins assay in domestic laboratories so as to provide suggestions for implementing the standardization and quality improvement. Methods: Two hundred and seventy-four laboratories those had developed or prepared to do anticoagulant proteins assay were selected from one thousand and five hundred participants in the national coagulation screening External Quality Assessment(EQA) program by an internet survey and then a questionnaire and quality control materials were sent to them to carry out a further survey. The questionnaire information was analyzed statistically. The results of quality control materials were grouped by the reagents and the average, median, standard deviation(s), coefficient variation(CV) of each group were calculated. The deviations or percentage deviations were determined by comparing the results of each laboratory to the target defined as the peer-group median after exclusion of outliers, and then the pass rates were calculated based on the criterion of RCPA, DGKL and the allowable total error based on biological variation. Results: Two hundred and thirty-five questionnaires were collected. The number of laboratories testing antithrombin(AT), protein C(PC) and protein S(PS) activity were 194, 63 and 50 respectively. The instruments and reagents were mainly from abroad (more than 96%), the matching rate of which were above 94%. For AT, PC and PS activity testing, there were 30.4%, 33.3%, 34.0% of laboratories did not perform verification assays respectively, and 8.8%, 7.9%, 14.0% of laboratories did not renew calibration curve when the reagent lots were changed. 11.3%, 17.5%, 16.0% of laboratories didn't run internal quality control, and 34.9%, 26.9%, 21.4% of laboratories only performed a single level of quality control. 4.1% of laboratories set the reference intervals of AT activity according to different age groups, and the percentage of that of PC and PS activity were 1.6% and 2.0%. 16.0% of laboratories set the reference interval of PS activity by sex. For normal control materials, the CV of AT, PC and PS activity results were 5.7%-12.9%, 4.2%-7.7% and 18.4%-33.1% while the CV for abnormal level were 13.3%-38.3%, 6.1%-14.4% and 31.5%-34.5% respectively. The pass rate was different when it was judged by different criteria. A suitable criterion for each item should be selected according to the concentration level of quality control materials. Conclusion: The comparability between laboratory results are not satisfactory and in order to promote quality improvement, it is necessary to develop guidelines, organize trainings and establish a national EQA scheme.

Standardization of haematology critical results management in adults: an International Council for Standardization in Haematology, ICSH, survey and recommendations.

INTRODUCTION: These recommendations are intended to develop a consensus in the previously published papers as to which parameters and what values should be considered critical. A practical guide on the standardization of critical results management in haematology laboratories would be beneficial as part of good laboratory and clinical practice and for use by laboratory-accrediting agencies. METHODS: A working group with members from Europe, America, Australasia and Asia was formed by International Council for Standardization in Haematology. A pattern of practice survey of 21 questions was distributed in 2014, and the data were collected electronically by Survey Monkey. The mode, or most commonly occurring value, was selected as the threshold for the upper and lower alert limits for critical results reporting. RESULTS: A total of 666 laboratories submitted data to this study and, of these, 499 submitted complete responses. Full blood count critical results alert thresholds, morphology findings that trigger critical result notification, critical results alert list, notification process and maintenance of critical results management protocol are described. This international survey provided a snapshot of the current practice worldwide and has identified the existence of considerable heterogeneity of critical results management. CONCLUSION: The recommendations in this study represent a consensus of good laboratory practice. They are intended to encourage the implementation of a standardized critical results management protocol in the laboratory.

[Evaluation and characterization of the certified reference materials for coagulation factor â § and â ¨ activity testing].

OBJECTIVE: To evaluate and characterize the certified reference materials for coagulation factor Ⅷ (FⅧ) and factor Ⅸ (FⅨ) activity testing. METHODS: The homogeneity and stability of three lots of certified reference materials (F01-F03) with different factor concentrations were evaluated according to guidelines"Reference materials-general and statistical principles for certification","Guidance on evaluating the homogeneity and stability of samples used for proficiency testing"and"Technical Norm of Primary Reference Material". The certified reference materials were characterized by eight laboratories using one-stage method, which were calibrated by the coagulation standard provided by the National Institute for Biological Standards and Control (NIBSC) in UK. RESULTS: The Coefficient of Variation (CV) of homogeneity test of FⅧ activity of three lots of certified reference materials were 3.9%, 3.3% and 3.4%, respectively. While that of FⅨ activity were 3.7%, 3.0% and 1.8%, respectively. The results of one-way ANOVA showed that all certified reference materials had good homogeneity (P>0.05), and the between-bottle homogeneity uncertainties (ubb) of FⅧ and FⅨ activity were 0.5%-2.9% and 0.1%-3.9%, respectively. All certified reference materials stored in -80 ℃ remained stable in 9 months by trend analysis, and the long-term stability uncertainties(ults) of FⅧ and FⅨ activity were 0.5%-5.1% and 1.3%-4.4%, respectively. The characterization uncertainties (uchar) of FⅧ and FⅨ activity testing were 0.9%-2.4% and 1.1%-2.4%, respectively. The combined uncertainties and extended uncertainties (coverage factor k=2) were calculated. The assigned values of each lot of certified reference materials for FⅧ activity were (85±13)%, (36.0±3.4)% and (20.5±2.3)%, and that were (102±13)%, (47.8±6.9)% and (29.3±3.8)% for FⅨ activity, respectively. CONCLUSIONS: The certified reference materials for FⅧ and FⅨ activity testing have good homogeneity and stability. The results of the characterization are accurate and reliable.

Sexual motivation is demasculinized, but not feminized, in prenatally stressed male rats.

Sexual motivation and copulation in male rats are associated with dopamine release in the nucleus accumbens. Demasculinized copulatory behavior has been demonstrated in prenatally stressed adult male rats. We have previously reported that approximately 80% of prenatally stressed male rats do not exhibit copulation and that no significant changes in nucleus accumbens dopamine release are seen during exposure to estrous females. In the present study, we investigated whether prenatal stress affects sexual motivation in these animals as adults. Pregnant Wistar rats were subjected to immobilization stress for two hours daily from day 15-19 of gestation. The prenatally stressed male offspring at the age of 3 months were allowed contact with receptive female rats for a 30 min period per week for 10 weeks; then, between the age of 5 and 6 months, their sexual motivation and copulatory activity were measured. Sexual motivation was measured in terms of sexual partner preference. The number of visits and the duration of each visit to an estrous female (stimulus female) or to a sexually active male rat (stimulus male) were recorded. Compared with control males, prenatally stressed male rats showed a significantly lower number of visits and a shorter duration of each visit to stimulus females. Prenatally stressed males showed no preference for male or female stimulus rats in terms of the number of visits and the duration of each visit, whereas control rats showed a significantly higher number of visits and duration of visits to female stimulus rats than male stimulus rats. A significant decrease in copulatory activity was observed in the prenatally stressed male offspring compared with control male rats, with most of the prenatally stressed males failing to show copulation. In vivo microdialysis experiments were performed on the nucleus accumbens with concurrent observation of sexual behavior. The prenatally stressed rats that did not exhibit copulation showed no significant changes in nucleus accumbens dopamine release during exposure to a stimulus male behind a wire-mesh barrier and the amount of dopamine release remained at the basal levels during actual physical contact. These results, combined with those of our previous report, indicate that sexual motivation in prenatally stressed male rats is demasculinized, but not feminized.

Enlarging effects of estradiol on the nuclear volume of neurons in the hypothalamus during aging.

Neuronal nuclear volumes (NNVs) were measured in the medial preoptic nucleus (MPN), anterior hypothalamic area (AHA) and arcuate nucleus (ARN) of young adult, middle-aged, and old rats of both sexes. The NNVs in the darkly stained sexual-dimorphic nucleus of the preoptic area (SDN-POA) and the lighter staining surrounding area (non-SDN-POA) within the MPN were measured separately. Intact young and middle-aged female rats had larger NNVs than those of the males in SDN-POA, non-SDN-POA and AHA but not in ARN. During aging, only intact old female rats manifested significant NNV shrinkage in all the measured areas. Long-term treatment with estradiol benzoate (EB) caused a significant enlargement of the NNVs in non-SDN-POA and ARN of middle-aged and old male rats as well as the NNVs in SDN-POA, non-SDN-POA and ARN of old female rats. The enlarging effect of EB on NNVs in both SDN-POA and non-SDN-POA of female rats could be prevented by ovariectomy. Furthermore, NNVs in SDN-POA and non-SDN-POA of ovariectomized female rats were even smaller than those of the age-matched intact female rats. These results indicate that: (1) the NNVs of MPN and ARN in male and female rats were enlarged after long-term exposure of physiological dose of estradiol; (2) the enlarging effects of EB on NNV in MPN can explain why the NNV of intact female rats is larger than that of males, and (3) during aging, the sex-specific shrinkage of NNVs in MPN, AHA and ARN of female rats may be due to an intrinsic aging process rather than long-term effects of EB.

Restoration of sexual behavior in aged male rats by intracerebral grafts of fetal preoptic area neurons.

A decline in sexual arousal and copulatory activity has been observed in male rats with advancing age. Grafting of fetal hypothalamic tissue into the third ventricle of aged male rats restores sexual behavior. This study investigated the effects of grafting fetal preoptic area (POA) neurons into the POA of aged male rats exhibiting decreased sexual behavior. We grafted suspensions of fetal POA neurons into the POA of 20 aged (19 to 24 months old) male rats that displayed no ejaculation. From 2 weeks after grafting, one or more series of male copulatory activity and sexual motivation tests were performed at intervals of 3 to 4 weeks. Three behavioral tests given 5 days apart were repeated for each animal in each series. Among the 20 aged rats with the fetal grafts, 15 showed improved sexual motivation, and 14 of these had copulatory activity restored to levels comparable with those of young males. Among these 14 rats, eight ejaculated during copulation. Copulatory behavior was restored between 21 and 45 days after grafting and persisted until the end of observation (2-4.5 months). Sexual performance did not improve in control aged male rats grafted with either fetal cerebral cortex neurons into the POA or POA, neurons into the ventromedial hypothalamus. The rats that received grafts of fetal POA neurons into the POA and recovered sexual performance also showed improvement or recovery to levels comparable to those in young males of serum testosterone concentrations, serum luteinizing hormone levels after castration, and the post-castration rise in luteinizing hormone. These results indicate that decreases in copulatory activity, sexual motivation, and some neuroendocrine functions in aged male rats are at least partially due to dysfunction of the POA.

The neonatal neurotoxicity of monosodium L-glutamate on the sexually dimorphic nucleus of the preoptic area in rats.

The neurotoxic effect of monosodium L-glutamate (MSG) on the morphologies in the darkly stained sexually dimorphic nucleus of the preoptic area (SDN-POA) and the lighter-staining surrounding area (non-SDN-POA) within the medial preoptic nucleus (MPN) was evaluated. Male and female Long-Evans rats were used. MSG (4 mg/g of body weight) was administered subcutaneously to pups on days 1 and 3 postnatally. Normal saline was used as the vehicle. At the age of 6 months, the rats were sacrificed and the brain tissues were fixed for histological examination. The morphological changes, i.e., total volume, density, total neuron number, neuronal nuclear volume (NNV) and ratio of pyknosis, of the SDN-POA and non-SDN-POA within the MPN, were estimated using the AMS VIDS III semiautomatic image-analytic system. The results indicate that neonatal MSG treatment caused significant neuronal loss and decreases in total volume of the SDN-POA and non-SDN-POA of male and female rats. However, only the SDN-POA of MSG-treated male rats showed a significant increase of pyknosis and decrease of neuronal density. A significant enlargement of NNV in the SDN-POA and non-SDN-POA was observed in the MSG-treated male rats. These results indicate that the MPN shows sex-specific and area-specific changes after neonatal neurotoxicity due to MSG.

Male sexual behavior is associated with LHRH neuron number in middle-aged rats.

LHRH administration is reported to facilitate male sexual behavior. The aim of the present study was to investigate whether male sexual behavior is associated with the number of LHRH neurons in the forebrain in middle-aged rats. Male Long-Evans rats (18-19 months) were assigned to three groups on the basis of sexual performance: (1) group MEI consisted of rats showing complete copulatory patterns, including mounts, intromissions and ejaculations, (2) group MI was composed of rats showing mounts and intromissions, but no ejaculation and (3) group NC were non-copulators, i.e. they did not show any copulatory behavior. Young adult rats (4-5 months), displaying sexual behavior, were used as controls. Following the sexual behavior tests, the number of LHRH neurons in the medial septum (MS), organum vasculosum of the lamina terminalis (OVLT), preoptic area (POA) and anterior hypothalamus (AH) was determined by immunocytochemistry. No difference was seen in the total number of LHRH neurons in these combined brain areas between group MIE and young controls. In the three middle-aged groups, the total number of LHRH neurons was greatest in group MIE, less in group MI, and lowest in group NC. In general, a similar trend was seen separately in the MS, OVLT and POA. These results suggest that changes in the number of LHRH neurons in the forebrain, in most cases, are age-related, at least in the middle-aged rats, but they also seem to be associated with male sexual performance.

Estradiol modulation of neuron loss in the medial division of medial preoptic nucleus in rats during aging.

The age-related morphological changes in the darkly stained sex-dimorphic nucleus (SDN-POA) and the lighter staining surrounding area (non-SDN-POA) within the medial division of preoptic nucleus of Long-Evans rats were studied. The long-term effects of estradiol benzoate (EB) on the changes were also assessed. During aging, the neuron loss in 14-(middle-age) and 22-month-old rats as well as increased pyknotic ratio of neurons in old male rats were observed in SDN-POA, but not in the non-SDN-POA. In female rats, significant neuron loss with advancing age was observed both in SDN-POA and the non-SDN-POA. Neuron loss in SDN-POA of EB-treated males was more severe than that of the intact males, while no significant difference of neuron loss was observed between EB-treated and age-matched intact female rats. However, neuron loss in SDN-POA of ovariectomized female rats was more severe than that of the age-matched intact females. These results indicate that age-related neuron loss in medial preoptic nucleus show sex-specific and area-specific features, and estradiol may play a important role in modulating neuron loss during aging.

Dopamine release in the nucleus accumbens during sexual behavior in prenatally stressed adult male rats.

In vivo microdialysis experiments were performed on the nucleus accumbens (NAc) during observation of sexual behavior (including motivation and copulation) to determine if there were any changes in NAc dopamine (DA) transmission in prenatally stressed (PS) adult male rats. Approximate 37% of control males and 83% of PS males did not exhibit copulation during the sexual behavior tests and no significant changes in NAc DA release were seen during exposure to estrous females. In contrast, both control and PS males that displayed copulatory behavior showed a marked increase in NAc DA release when presented with a sexually receptive female behind a screen and this increased further during actual copulation. The increase in DA release in copulatory PS males was not significantly different from that in sexually active control males. In addition, a similar extent in DA release induced by high potassium perfusate was observed in all rats. These results suggest that prenatal stress may result in a deficit in DA neurotransmission in the NAc and this deficit may possibly cause impaired male sexual behavior in rats.

Effects of age on dopamine release in the nucleus accumbens and amphetamine-induced locomotor activity in rats.

The effects of age on dopamine (DA) release in the nucleus accumbens (NAc) and on amphetamine (AMPH)-induced locomotor activity were studied by microdialysis in freely-moving young (5 month) and old (24 month) rats. Both basal extracellular DA and 3,4-dihydroxyphenylacetic acid (DOPAC) release and that following intra-accumbens perfusion of AMPH (1-10 microM) were significantly lower in old rats. After intraperitoneal injection of AMPH (1.5 mg/kg), no age-related change in DA release was seen in the NAc, but locomotor activity was found to increase much more in young rats than in old ones. These results indicate that (1) old rats show decreased extracellular DA and DOPAC release, both in the basal state and following intra-accumbens infusion of AMPH, and (2) the age-related locomotor activity induced by systemic injection of AMPH is not paralleled by changes in DA release in the NAc.