The paper is to report the study of pharmacokinetics of transdermal administered nicotine in the brain of freely moving rat by using microdialysis with stable labeled isotope as internal standard. The pharmacokinetic behavior of nicotine in Sprague Dawley rat brain was investigated after intranasal administration (3.75 mg). Brain fluid samples were collected by intracerebral microdialysis with DL-nicotine as internal standard. Concentrations of nicotine and DL-nicotine in the sample were measured by HPLC-MS/MS. Main pharmacokinetic parameters were calculated and analyzed by Das 2.0 pharmacokinetic software. The recovery of nicotine and the delivery of DL-nicotine were the same. The fate of absorption and distribution was two compartment model and the values of t1/2alpha was 170.31 min, t1/2beta was 263.30 min and the AUC(0-infinity) was 2.75 x 10(5) microg x L(-1) min separately. DL-nicotine can be used to calibrate the recovery of nicotine, and the new method of stable isotope microdialysis can be used to study the pharmacokinetics of freely moving rat. It will make sense for the treatment of addiction of tobacco and provide a new thought for the research of pharmacokinetics-pharmacodynamic combination.

Objective To establish a stable animal model for sequentially dynamic and direct monitoring of the skin wound infection. Methods The mice with full-thickness skin incisions were replicated. After immediate subcutaneous suture,the mice were randomly divided into four groups,ie,Group A was inoculated with 50 μl sterile PBS solution),Groups B,C and D were inoculated with 50 μl suspension containing 1 × 106,1 × 108 and 1 × 1010 colony forming unit (CFU)/ml bioluminescent methicillin-resistant staphylococcus aureus (MRSA) respectively.Then,the diet behavior of each group was observed and the mean weight and mortality of each group were also recorded at different time points.The bioluminescent intensity of fluoresce in the wounds was recorded at different time points by using the charge-coupled device (CCD) based imaging system.Local wound tissues were incised at 24 hours after inoculation for HE staining so as to observe wound inflammatory reaction in each group.Wound healing time of each group was also recorded. Results ( 1 ) Average weight:Groups A and B showed unobvious changes in weight; Group C lightened until day 3 after inoculation and then recovered gradually to the preinoculation level at day 14; Group D lightened gradually until death.(2)Mortality:Groups A and B had no death; Group C had 10％ deaths at day 14; Group D had 100％ deaths.(3) Bioluminescent intensity of wounds:Groups A and B showed a gradual weakened luminescence since the day of inoculation and had almost complete disappearance at days 5 and 7 respectively; there was no sign of obvious increase or decrease in Group C from the day of inoculation till day 14 ; Group D had a gradual increase since the day of inoculation and the luminous area expanded until the death.(4) HE staining at 24 hours after inoculation:all the four groups showed inflammatory cell infiltration,especially in Groups C and D.(5) Wound healing time:wound healed at days 5 and 7 after inoculation in Groups A and B; the wounds showed no healing even at day 14 in the Group C,but the wounds length and area did not show obvious enlargement or diminishment ; the wounds extended gradually until the death in the Group D,since the day of inoculation. Conclusions The inoculation of 50 μl suspension with 1 × 108 CFU/ml bioluminescent MRSA to full-thickness skin incision rats allows direct,real-time dynamic and continuous detection of the occurrence and development of the wound infections.The infection model is easy to make and has stability and high repeatability.

Background The incidence of dry eye is gradually increased,and researches showed that inflammation participated in the pathogenesis and development of dry eye.The current therapy for dry eye can only relieve symptom but not achieve final cure.Stem cell therapy has been used in the treatment of limbal stem cell deficiency.However,whether it is feasible for the stem cell treating dry eye is still unclear.Objective This study attempted to investigate a new approach to treat dry eye syndrom by using human umbilical cord mesenchymal stem cells (HUCMSCs).Methods The use and care of experimental animals complied with the Tsinghua University School of Medicine Laboratory Animal Care Details.HUCMSCs were cultured and cell suspension was prepared with the cell density of 5×105/ml.Twenty 24-week-old male NOD/Ltj mice were randomized into 4 groups.0.1 ml PBSHUCMSCs suspension was injected via tail vein or lacrimal respectively in the caudal vein injection group and lacrimal injection group,and 10 μ1 PBS-HUCMSCs suspension was topically administed in the eye drops group.The NOD/Ltj mice without any treatment served as the model group.Five male ICR mice were used as the normal control group.Tear secretion was quantitatively detected with phenol red cotton thread in 1,2,3 weeks after injection,and corneal epithelial defect was scored by fluorescein staining.The serum levels of interleukin (IL)-6,IL-17a,tumor necrosis factor-α (TNF-αt) and interferon-γ (IFN-γ) were assayed by ELISA.The relative expression levels of p65,Stat3,Stat5 and extracellular signal-regulated kinases (Erk)-1 in lacrimal gland were detected by Western blot.Results The tear secretion amount was significantly different among the model group,caudal vein injection group,lacrimal injection group and eye drops group in various time points (1 week:F =3.700,P =0.040;2 weeks:F =5.150,P =0.008;3 weeks:F=10.130,P＜0.001).The tear secretion amount was increased in the caudal vein injection group and lacrimal injection group compared with the model group in different time points (all at P＜0.05),and no significant difference was seen in tear secretion amount between eye drops group and model group among various time points (all at P＞0.05).The fluorescein staining score was 3.00±0.63,9.40±1.62,5.20±1.17,4.20±1.17 and 7.20±0.98 in the ICR mouse control group,model group,caudal vein injection group,lacrimal injection group and eye drops group 1 week after injection respectively,and the scores were significantly lower in the caudal vein injection group,lacrimal injection group and eye drops group than those in the model group (P =0.001,0.000,0.033).The serum levels of IL-6,IL-17a and TNF-α in the caudal vein injection group were evidently lower than those in the model group (t =4.70,3.46,11.0,all at P＜0.01),but no significant difference was displayed in the serum IFN-γ level among the five groups (F=1.740,P=0.170).The expressions of STAT5 were significantly decreased in the mice treated with tail vein injection and lacrimal injection compared with mice without treatment (both at P＜0.05).Conclusions Administration of HUCMSCs via intravenous and lacrimal injection can alleviate the inflammatory response during progression of dry eye syndrome by down-regulating the serum level and expression of inflammation-related factors in NOD/Ltj mice.The topical administration of HUCMSCs eye drops can attenuate the symptom of dry eyes by lubricating the cornea and suppling nutrition.

Objective To investigate the clinical manifestations of early postoperative internal hernia. Methods Patients who were diagnosed with early postoperative small bowel obstruction(EPSBO)within 30 days after operation and underwent laparotomy between 1994 and 2006 were included for study.Clinical and radiological findings were analyzed. Results Totally 38 EPSBO patients were identined.among those,9 patients(23.7%)had an internal hera ag the cause of the howel obstruction.Other causes included intestinal adhesions in 27 patients(71.1%),gallstone ileus in 1 patient(2.6%)and stoma obstruction in 1 patient(2.6%).In the internal hernia group,6 cases were male and 3 cases were female witIl a mean age of 53.6 years.The mean time from the primary operation to symptom development was 7.8 d(range,2～17 d)and the mean time of conservative treatment Was 3.4 d(range,1～8 d).The main clinical features included:complete mechanical obstruction with symptoms rapidly progressing and early bowel strangulation.Specific radiologic abnormalities misht be identified,especially by contrast-enhanced CT.In this series,intestinal strangulation was found in 6 patients with bowel necrosis in 4 eases,necessitating howel resection in 5 patients.Wound infection developed in one cage and there was no perioperative death.Conclusion Internal hernia can occur early postoperatively and it bears a high risk of strangulation and bowel necrosis.Prompt operative intervention should be carried out in highly suspicious patients in order to avoid complications and achieve good outcome.

Objective To study the influence of traction on the blood circulation of femur head and its evaluation by DSA. Methods Using micro-catheter, transfemoral selective femoral circumflex arteriography in 22 healthy dogs was performed in unilateral hip before (Group A,n =22) and immediately (Group B,n=22) ,30( Group D,n =22) ,60(Group E,n=20) ,90( Group F,n = 10),120 (Group G,n=10) minutes during 2 kg skin hip traction, and immediately after traction removal (Group H,I,J,L and 0), and 30 minutes after traction removal with 60,90 and 120 minutes continuous traction(Group K,M and P),and 60 minutes after traction removal with 90 and 120 minutes continuous traction(Group N and Q). DSA was also performed immediately during 4 kg weight traction before continuous traction in 12 hips( Group C).Blood circulation of the femoral head was evaluated mainly by observing its perfusion and time of circulation. Femur head perfusion was assessed as good scoring 3,poor scoring 2 and extremely poor scoring 1. Femur head circulation time was assessed as normal scoring 3 .prolonged scoring 2 and remarkably prolonged scoring 1. Analysis of variance was employed for analysis of the angiographic findings between different groups.Results Good femoral head perfusion in Group A to Q was 22,0,0,0,0,0,0,22,22,1,18,0,0,8,0,0 and 1 hips respectively, poor one was 0,22,8,22,15,4,1,0,0,15,2,4,6,2,1,3 and 8 hips, respectively,extremely poor one was 0,0,4,0,5,6,9,0,0,4,0,6,4,0,9,7 and 1 hips, respectively; and normal femoral head blood circulation time was 22,0,0,0,0,0,0,22,22,1,18,0,0,8,0,0 and 1 hips, respectively, prolonged one was 0,22,9,22,15,4,2,0,0, 15,2,5,7,2,2,4 and 8 hips, respectively, remarkably prolonged one was 0,0,3,0,5,6,8,0,0,4,0,5,3,0,8,6 and 1 hips, respectively. F value of femoral head perfusion among group A and B,group B and C,group B,D,E,F and G,Group H,I,J,L and O,group K,M and P,Group N and Q was 437. 48,30. 25,29. 04,132. 69,143. 73,25.20, respectively, and their P value was all <0. 01. F value of femoral head circulation time among group A and B,group B and C,group B,D,E,F and G,Group H,I,J,L and O,group K,M and P,Group N and Q was 386. 26,31. 83,22.43,141. 94,119.69,21.68, respectively, and their P value was all < 0.01. Conclusions Traction could lead to ischemic response and circulation disorder of canine femoral head. The longer the traction time or the bigger the traction weight was, the poorer the femoral head perfusion and the longer the femoral head circulation time were, and the slower they recovered. DSA could directly reflect these changes in the femoral head with these angioarchitectural and hemodynamic indexes.

OBJECTIVETo explore the feasibility of microdialysis techniqiue to be used in pharmacokinetic study of Chinese medicine, taking Shuanghuanglian as a model drug.

METHODThe samples were obtained by retrodialysis, determined by HPLC gradient elution to calculate the in vitro recovery rate (RR) of specific components. To study the difference of RR of a certain component in different dialysis mediums, and the effect of flow rates and concentration on RR.

RESULTAlong with the increase of number of substances in the dialysis medium, the RR of specific components reduced. But the RR was independent of the concentration of the component in the dialysis medium. The RR reduced with the increasing flow rate in the same dialysis medium.

CONCLUSIONThe microdialysis technique can be used in pharmacokinetics study of Chinese medicine.

Calibration ; Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal ; analysis ; isolation & purification ; pharmacokinetics ; Feasibility Studies ; Microdialysis ; methods

OBJECTIVEUsing the stable isotopes as the internal standard of microdialysis technology to establish a new method to study the whole and local brain dynamics of nicotine percutaneous preparations.

METHODUsing th healthy rats as experimental animals, administrating nicotine in abdominal transdermal way, then sample in the blood and brain simultaneously by microdialysis which use deuterium nicotine (DL-nicotine) as internal standard. Detecting the samples by LC-MS/MS method.

RESULTThe configuration process in blood and brain both conforms to 2 compartments model, t1/2 is 29.38 min, t1/2beta is 208.51 min, AUC(0-infinity) is 152 127.10 microg x min x L(-1) in the blood t1/2 is 86.64 min, t1/2beta is 386.00 min, AUC(0-infinity) is 152 820.90 microg x min x L(-1) in the brain.

CONCLUSIONDl-nicotine can be used as internal standard of nicotine to correcte the recovery; Stable isotopes internal standard microdialysis technology can be used for studing the whole and the local pharmacokinetic of nicotine and also provide new ideas and methods to studing the process of new drug delivery system.

Animals ; Brain ; metabolism ; Brain Chemistry ; Deuterium ; chemistry ; Isotope Labeling ; methods ; Male ; Microdialysis ; methods ; Nicotine ; blood ; pharmacokinetics ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo study the relationship between neuropeptide Y (NPY) and diabetes by examining the content and distribution of NPY and its mRNA expression in the hypothalamus and pancreas of STZ-diabetic rats.

METHODSThirty Wistar rats were randomly divided into 3 groups (diabetic group, diabetic insulin treatment group, and control group). After feeding for 24 weeks, the rats were sacrificed. The expression of NPY in the hypothalamus and pancreas was detected with immunohistochemistry and in situ hybridization.

RESULTS(1) The hypothalamic content of NPY and its mRNA were significantly increased in STZ-diabetic rats in comparison with normal controls. Increased expression of NPY mRNA was found only in the arcuate nucleus and not in the paraventricular nucleus in diabetic rats, suggesting that NPY was produced in the arcuate nucleus. (2) The hypothalamic content of NPY and its mRNA in STZ-diabetic rats were visibly reduced after insulin treatment compared with that in untreated diabetic rats. This supports the hypothesis that insulin deficiency in the brain may be responsible for increased hypothalamic NPY gene expression in diabetic rats. (3) The increase of hypothalamic NPY in STZ diabetic rats associated with hyperphagia and polydipsia could be reversed by insulin replacement, suggesting that increased hypothalamic NPY contributes to the pathophysiological progress of the diabetic state. (4) The present study demonstrated for the first time that the content of NPY and its mRNA in the pancreas was increased in STZ-diabetic rats, and that the distribution of NPY-positive cell in islets was changed from the periphery to the whole islet. The content and distribution of NPY and its mRNA in islets were not changed by insulin treatment.

CONCLUSIONIncreased NPY in the hypothalamus results in hypophagia and polydipsia, while the implication of increased NPY in the pancreas of diabetic rats is not clear.

Animals ; Blood Glucose ; drug effects ; Diabetes Mellitus, Experimental ; drug therapy ; genetics ; metabolism ; Drinking ; drug effects ; Eating ; drug effects ; Female ; Gene Expression Regulation ; drug effects ; Hypothalamus ; drug effects ; metabolism ; Immunohistochemistry ; In Situ Hybridization ; Insulin ; therapeutic use ; Neuropeptide Y ; drug effects ; genetics ; metabolism ; Pancreas ; drug effects ; metabolism ; RNA, Messenger ; drug effects ; genetics ; metabolism ; Rats ; Rats, Wistar

OBJECTIVETo investigate the association of Pro12Ala variant in peroxisome proliferator-activated receptor-gamma2 gene with type 2 diabetes mellitus and its clinical characteristics.

METHODSThe genotypes of Pro12Ala variant in peroxisome proliferator-activated receptor-gamma2 gene were determined by polymerase chain reaction-restriction fragment length polymorphisms assay in 401 unrelated subjects of the Han population in the southern part of China (including 180 subjects with normal glucose tolerance and 221 type 2 diabetic patients). The clinical data were also analyzed.

RESULTSThe allele frequencies in the case and control groups were 96.15%,96.11% for P and 3.85%, 3.89% for A; the genotype frequencies were 92.77%, 92.22% for PP, 6.78%, 7.78% for PA and 0.45%, 0 for AA. The Pro12Ala variant of peroxisome proliferator-activated receptor-gamma2 was not significantly associated with type 2 diabetes. The Pro12Ala polymorphism of peroxisome proliferator-activated receptor-gamma2 in diabetes patients was associated with increased waist circumference and waist to hip ratio. The Pro12Ala polymorphism in Chinese population was similar to that in Japanese population and was different from that in European and American population.

CONCLUSIONThe above data showed that the Pro12Ala variant of peroxisome proliferator-activated receptor-gamma2 was not significantly associated with type 2 diabetes, but it could be associated with abdominal obesity in type 2 diabetes. The significant difference of Pro12Ala of peroxisome proliferator-activated receptor-gamma2 among various races was observed.

Adult ; Alanine ; genetics ; Alleles ; Amino Acid Substitution ; Blood Glucose ; metabolism ; Body Constitution ; Body Mass Index ; Cholesterol ; blood ; Cholesterol, HDL ; blood ; Cholesterol, LDL ; blood ; Diabetes Mellitus, Type 2 ; blood ; genetics ; pathology ; Female ; Gene Frequency ; Genotype ; Humans ; Insulin ; blood ; Male ; Middle Aged ; Proline ; genetics ; Receptors, Cytoplasmic and Nuclear ; genetics ; Transcription Factors ; genetics ; Triglycerides ; blood