Expression of hypoxia inducible factor-1 alpha and ischemic erythropoietin tolerance in the brain of cerebral ischemic tolerance model rats

BACKGROUND： Hypoxia inducible factor-1 alpha （HIF-1 （x） and erythropoietin（EPO）, possessing neuroprotective effect in the cerebral ischemia, might play an important role in the formation of cerebral ischemic tolerance （IT）. OBJECTIVE：To observe the neuroprotective effect of cerebral ischemic preconditioning（IPC） of rats, and the expression and mechanism of HIF-1α and target gene erythropoietin in the brain tissue following the formation of cerebral IT. DESIGN ： A randomized and controlled observation SETTING： Department of Neurology, the Affiliated Hospital of Medical College, Qingdao University MATERIALS： Totally 84 enrolled adult healthy male Wistar rats of clean grade, weighing 250 to 300 g, were provided by the Animal Experimental Department, Tongji Medical College of Huazhong University of Science and Technology. Ready-to-use SABC reagent kit and rabbit anti-rat HIF-1α monoclonal antibody were purchased from Boshide Bioengineering Co.Ltd （Wuhan）; Rabbit anti-rat EPO monoclonal antibody was purchased from Santa Cruz Company （USA）. METHODS： This experiment was carried out in the Department of Anatomy, Medical College, Qingdao University during March 2005 to March 2006. ① The 84 rats were divided into 3 groups by a lot： IPC group （n=40）, sham-operation group （n=40） and control group （n=4）. In the IPC group, middle cerebral artery was occluded for 2 hours respectively on the 1^st, 3^rd, 7^th, 14^th and 21^st days of the reperfusion following 10-minute preischemia was made using a modified middle cerebral artery second suture method from Zea-Longa. The rats were sacrificed 22 hours after reperfusion in the end of middle cerebral artery occlusion （MCAO）. That was to say, after 10-minute preischemia, suture was exited to the extemal carotid artery and embedded subcutaneously. Middle cerebral artery was occluded again to form the second reperfusion at the set time point after reperfusion. Twenty-two hours later, rats were sacrificed; In the sham-operation group,the preischemia was substituted by sham-operation（only common carotid artery and crotch were exposed, and MCAO by suture was omitted）, and the other procedures were the same as those in the IPC group. In the control group, rats were given sham-operation twice at an interval of one day, and they were sacrificed 24 hours after the second sham-operation. ② Brain tissue was taken from the rats in each group. Cerebral infarction area of each layer was measured with TTC staining, and total cerebral infarction volume （The total cerebral infarction area of each layerxinterspace ） was calculated. After brain tissue was stained by haematoxylin-esoin （HE）, the form of nerve cells was observed under an optical microscope, and the expressions of HIF-1α（and EPO protein in the brain tissue were detected with immunohistochemical method. MAIN OUTCOME MEASURES： ①Cerebral infarction volume;②form of nerve cell; ③ the expression of HIF-1α and EPO protein in the brain tissue. RESULTS：Totally 84 rats were enrolled in the experiment. The dead rats were randomly supplied during the experiment, and finally 84 rats entered the stage of result analysis. ① Detection of cerebral infarction volume of rats in each group： Cerebral infarction volume in the IPC group was significantly smaller than that in the sham-operation group on the 1^st, 3^rd and 7^th days after reperfusion respectively [（161.2±6.9） mm^3 vs （219.9±11.2） mm^3, （134.9±9.0） mm^3 vs （218.6±13.0） mm^3, （142.9±13.7） mm^3 vs （221.3±14.2） mm^3, t=-8.924, 10.587,7.947, P〈 0.01]. ② Observation of nerve cell form of brain tissue： HE staining showed that the ischemic degree, range and cerebral edema degree of IPC group were significantly milder than those of sham-operation group. ③ The expressions of HIF-1α and EPO protein in cerebral cortex and hippocampus ： The expression of HIF-1αof IPC group was significantly higher than that of sham-operation group on the 1^st, 3^rd and 7^th days after reperfusion respectively （125.93±3.79 vs 117.65±5.60, 140.63±4.64 vs 119.33±4.26, 131.15±2.74 vs 107.60±3.89, t=2.449, 6.763,9.899,P 〈 0.05-0.01）. The expression of EPO of IPC group was significantly higher than that of sham-operation group on the 3^rd and 7^th days after perfusion respectively （141.68±3.29 vs 126.33±4.51, 138.88±2.59 vs 125.58±6.18,t=5.499,3.970, P〈 0.05）. CONCLUSION ： ①IPC can protect the never cells in rat brain and the best time to onset of cerebral IT induced by IPC is 1 to 7 days after reperfusion. ② Neuroprotective effect of cerebral IT might be related to the expression of HIF-1α and its target gene EPO.

Expression of calbindin D28K in substantia nigra of model rats with Parkinson disease

BACKGROUND： Previous researches suggested that expression level of calbindin D28K mRNA decreased in substantia nigra （SN） of model rats with Parkinson disease （PD）, and this might be related to the decrease of anti-degeneration potentials of dopaminergic neurons. OBJECTIVE： To observe expression changes of calbindin D28K in SN dopaminergic neurons during their degeneration and death in midbrain of PD model rats. DESIGN： Completely randomized grouping design SETTING： Department of Neurobiology, Xuzhou Medical College MATERIALS＂ A total of 92 healthy male SD rats, with the age of 3 months, weighing 200-250 g, were selected from Experimental Animal Center of Xuzhou Medical College [certification： SCXK （su） 2003-0003]. Calbindin D28K（CB）, tyroxine hydroxylase （TH）, ABC kit, 6-hydroxydopamine （6-OHDA） and Nissl dyes were provided by Sigma Company, and sheep serum was provided by Beijing Zhongshan Company. METHODS： The experiment was carried out in the Neurobiological Center of Xuzhou Medical College from October 2003 to October 2004. ① With lot method, rats were divided into blank control group （n=28）, experimental control group （n=-28） and experimental group （n=36）. Rats in experimental group were injected with 6-OHDA at right corpus striatum for PD modeling; rats in experimental control group were injected with saline at the same site; rats in blank control group did not give any injections.② On the 7^th, 14^th, 21^st and 28^th days, SN segments on right midbrain from every 5 rats in experimental group were fixed, embedded with paraffin and cut into successively coronary pieces. Rats in other two groups were treated with the same methods and then stained with Nissl to show neuronal form. Meanwhile, CB and TH antibodies staining with immunohistochemistry were used to show CB containing dopaminergic neurons and dopaminergic neurons, and cells were calculated and observed under optic microscope. ③ On the 14^th and 28^th days, every 4 rats in experimental group and every 4 rats in control group were selected to obtain their brains and separate SN on the injured side. Western blot was used to detect expression of calbindin D28K, protein band was scanned with imaging equipment, and data were analyzed with LabWorks software. MAIN OUTCOME MEASURES：① On the 7^th, 14^th, 21^st and 28^th days, Nissl staining results of SN neurons and immunohistochemical staining results of CB and TH antibodies; ② On the 14^th and 28^th days, Western blot results of calbindin D28K in SN neurons, RESULTS; Among 92 rats, 2 rats in experimental group died after 1 day due to 6-OHDA injection and other 90 rats were involved in the final analysis. ①Nissl staining results： On the 7^th day of 6-OHDA injection, most neuronal somas on right SN pars compacta were shown as deep pycnosis or lysis breakage; on the 14^th and 21^st days, amount of neurons was decreased remarkably; on the 28^th day, most neurons in SN pars compacta disappeared. ② Results of immunohistochemical staining： Amount of positive neurons of calbindin D28K in right SN pars compacta was not changed on the 7^th day after 6-OHDA injection; on the 14^th day, the amount was higher in experimental group than that in both control groups （P 〈 0.01） and was decreased till the 21^st day, but it was still higher than that in the two control groups （P 〈 0.05）; on the 28^th day, positive neurons of calbindin D28K nearly disappeared, and the amount was lower than that in the two control groups （P 〈 0.01）. In addition, on the 7^th day after 6-OHDA injection into corpus striatum, positive TH neurons decreased 24% in right SN, and there was significant difference from that in control groups; on the 14^th, 21^st and 28^th days, positive TH neurons decreased 37%, 46% and 64%, respectively. ③ Western blot results： On the 14＇h day after 6-OHDA injection into corpus striatum, expression of calbindin D28K in right SN was higher in experimental group than that in both control groups （P 〈 0.05）; however, on the 28^th day, the expression was lower than that in the two control groups （P 〈 0.01 ）. CONCLUSION ： During degeneration and death of dopaminergic neurons, CB expression in SN pars compacta increases firstly and decreased gradually.

Effects of Chuanxiongqin hydrochloride on increasing the fluidity of brain cell membrane and scavenging free radicals in model rats with ischemia/reperfusion injury

BACKGROUND: The fluidity of cell membrane can be affected by various factors. Many experiments have confirmed that the ischemia/reperfusion of organic tissue can increase the contents of free radicals, which lead to high rigidity and low fluidity of cell membrane, and the conditions can be changed by Chuanxiongqin. OBJECTIVE: To observe the effect and mechanism of Chuanxiongqin hydrochloride on the fluidity of brain cell membrane in rat models of ischemia/reperfusion. DESIGN: A completely randomized controlled animal trial. SETTINGS: Institute of Brain Sciences; Department of Physiology, Medical College, Datong University. MATERIALS: Twenty male grade Ⅰ Wistar rats of 170-220 g were randomly divided into model group (n =10) and control group (n =10). Chuanxiongqin hydrochloride (molecular mass was 172.2) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (batch number: 0817-9803); Spin labelers: 5-doxyl-stearlic acid methylester (5DS), 16-doxyl-stearlic acid methylester (16DS), xanthine, xanthine oxidase (XOD) and 5,5-dimeth-1-pyrroline- N-oxide (DMPO) from Sigma Company; Bruker ESP 300 electron paramagnetic resonance (EPR) spectrometer by Bruker Company (Germany). METHODS: The experiments were carried out in the State Key Laboratory of Natural and Biomimetic Drugs, Peking University from June 2001 to July 2002. In the model group, rats were made into models of cerebral ischemia by 30-minute ligation and 2-hour reperfusion of common carotid arteries; The rats in the control group were not made into models. The order parameter (S) and rotational correlation time (τc) were detected with the ESR spectrometer by means of spin labeling. The greater the S and τc, the smaller the fluidity. Meanwhile, the clearance rate of free radicals was detected with ESR spin trapping. The measurement data were compared using the t test. MAIN OUTCOME MEASURES: The S, τc and clearance rates of O2 · and OH· free radicals were compared between the model group and control group. RESULTS: The S and τc in the model group [0.738 4±0.003 5; (8.472±0.027)×10-10 s/circle] were obviously different from those in the control group [0.683 9±0.008 3; (7.945±0.082)×10-10 s/circle, t =5.731, 5.918, P < 0.05], which suggested that ischemia/reperfusion injury decreased the fluidity of brain cell membrane. After adding Chuanxiongqin hydrochloride, there were no obvious differences between the model group [0.688 5±0.030 5; (7.886±0.341)×10-10 s/circle] and control group (P > 0.05), indicating that Chuanxiongqin hydrochloride could recover the fluidity of brain cell membrane after ischemia/reperfusion injury close to the level in the normal control group. Chuanxiongqin hydrochloride could directly scavenge the O2 · and OH· free radicals, and the maximal clearance rates were 83.92% and 44.99% respectively. CONCLUSION: Chuanxiongqin hydrochloride increases the fluidity of membrane of ischemia-injured brain cell by scavenging both O2 ·and OH· free radicals.

Preparation of 6-[^(18)F]fluoro-L-DOPA and its biodistribution in normal and unilateral PD model rats

No-carrier-added 6-[^18F] fluoro-L-DOPA(6-FDOPA) was synthesized via a multistep procedure from a commercial available precursor,6-nitroveratraldehyde,The total synthesis time was 75min,with a radiochemical yield of (10±3)%,high radiochemical purity(>99%) and high enantiomeric purity(>95%).The biodistributions of 6-FDOPA in normal and unilateral PD model rats were measured.The results from normal rats showed the expected high concentration of radioactivity in striatum and low distrbutions in cerebrum,cortex and cerebellum.The ration of the radioactivity in striatum to cerebellum reached a peak value(5.9) at 60 min.In unilateral PD model rate.whose substania nigra of the right side had been damaged by pre-treated with 6-OHDA,the radioactive concentration in striatum of the damaged side was significantly lower than that of the undamaged side or that of both sides in striatum of control groups.

Rat models of frozen shoulder:Classification and evaluation

Frozen shoulder(FS),also known as adhesive capsulitis,is a condition that causes contraction and stiffness of the shoulder joint capsule.The main symptoms are per-sistent shoulder pain and a limited range of motion in all directions.These symp-toms and poor prognosis affect people's physical health and quality of life.Currently,the specific mechanisms of FS remain unclear,and there is variability in treatment methods and their efficacy.Additionally,the early symptoms of FS are difficult to distinguish from those of other shoulder diseases,complicating early diagnosis and treatment.Therefore,it is necessary to develop and utilize animal models to under-stand the pathogenesis of FS and to explore treatment strategies,providing insights into the prevention and treatment of human FS.This paper reviews the rat models available for FS research,including external immobilization models,surgical internal immobilization models,injection modeling models,and endocrine modeling models.It introduces the basic procedures for these models and compares and analyzes the advantages,disadvantages,and applicability of each modeling method.Finally,our paper summarizes the common methods for evaluating FS rat models.

The bio-active components of the Mongolian medicine Horcha-6 and therapeutic mechanism in the rat migraine model

Background:The active components of Horcha-6 were identified using liquid chromatography with tandem mass spectrometry.Also,we investigated the potential mechanisms that explain why Horcha-6 may be effective in treating migraines through the use of network pharmacology and a rat migraine model.Methods:After identifying the active components of Horcha-6,the corresponding genes of the active components’target were obtained from the Universal Protein database,and a“compound-target-disease”network was constructed using Cytoscape 3.9.0 software.For the in vivo experiments,nitroglycerin was injected intraperitoneally into rats to create a migraine model.Pre-treatment with Horcha-6 was administered orally for 14 days,and rats were subjected to migraine-related behavior tests.RNA sequencing was performed to identify the gene expression regulated by Horcha-6 in the trigeminal nerve.Results:A total of 903 chemical components of Horcha-6 have been collected in the liquid chromatography with tandem mass spectrometry.We discovered 55 of the Horcha-6 bio-active components that were evaluated based on their Percent Human Oral Absorption(≥30%)and DL values(≥0.185)on the traditional Chinese medicine systems pharmacology database.The“compound-target-disease”network contained 163 intersection targets with the migraine state.Gene Ontology analysis indicated that these components significantly regulated the immune response,vascular function,oxidative stress,etc.When Kyoto Encyclopedia of Genes and Genomes enrichment analysis was performed,we observed that most of the target genes were significantly enriched in the inflammation and neuro-related signaling pathway,toll-like receptor signaling pathway,neuroactive ligand-receptor interaction,etc.These predictions were further demonstrated via in vivo animal model experiments.The RNA sequencing results showed that 41 genes were down-regulated(P<0.05)and 86 genes were up-regulated(P<0.05)in the Horcha-6 treated group compared with the untreated group.Those genes were mainly involved in neuromodulation,vascular function,and hormone metabolism.Conclusion:The 55 bio-active components in Horcha-6 regulate inflammation,hormone metabolism,and neurotransmitters and have potential as a therapy to treat migraines.

Effects of electroacupuncture at lower he-sea points on interleukin-1β and high mobility group box 1 in model rats with ulcerative colitis

Objective To comparatively observe the effect of electroacupuncture at digestive system-related lower he-sea points on the expressions of serum interleukin-1β（IL-1 β）, tumor necrosis factor-α（TNF-α） of colon tissues and high mobility group box 1 protein（HMGB 1） of ulcerative colitis（UC） model rats, and to explore whether there is relative specificity of electroacupuncture at Shàngjùxū（上巨虚 ST 37）, one of lower he-sea points of large intestine, in treatment of bowel diseases. Method A total of 60 SD rats were randomly divided into control group, model group, ST 37 group, Zúsānl？（足三里 ST 36） group, Xiàjùxū（下巨虚 ST 39） group and Yánglíngquán（阳陵泉 GB 34） group. There were ten rats in each group; five were males, and five were females. UC models were established by clysis with 2, 4, 6-trinitrobenzene sulfonic acid/alcohol solution. After modeling, treatment was conducted for ten days, specimens were collected, colonic ulcers and inflammation were inspected visually and scored. The content of serum IL-1β and the expressions of TNF-α and HMGB 1 in colon were detected through ELISA. Results 1 Compared with control group, the scores of colonic ulcers and inflammation, the content of serum IL-1β and the expressions of TNF-α（except ST 37 group） and HMGB 1 were all higher（P〈0.05, P〈0.01）; 2 compared with model group, the scores of colonic ulcers in ST 36 group and ST 37 group were lower obviously（P〈0.05, P〈0.01）; the expressions of IL-1β, TNF-α and HMGB 1 in the four treatment groups were lower obviously（P〈0.01）; 3 compared with ST 37 group, the expressions of IL-1β, TNF-α and HMGB 1 in other three treatment groups were higher obviously（P〈0.05, P〈0.01）; and the scores of colonic ulcers in ST 39 group and GB 34 group were higher obviously（P〈0.05）. Conclusion 1 The score of colonic ulcers can be reduced through electroacupuncture at ST 37, ST 36, ST 39 and GB 34, which can also reduce the content of serum IL-1β and the expressions of TNF-α and HMGB 1, and effectively inhibit inflammatory response of colon caused by UC; 2 the effect trend of the four acupoints in treatment of UC is： ST 37ST 36ST 39GB 34, and electroacupuncture at ST 37 has the best effect with relative specificity.

A Model of Focal Cortical Infarctionin Rat:Mini mally Invasive Craniotomy

目的建立一种稳定的大鼠脑局灶性梗死模型,以用于脑梗死后神经干细胞移植的长期观察。方法37只大鼠随机分为实验组和对照组。微创法经颞骨局部钻孔开颅,采用直接结扎大脑中动脉终段,同时永久结扎同侧颈总动脉、暂时性夹闭对侧颈总动脉的方法制备大鼠脑梗死模型。通过大鼠脑梗死后的神经功能评分、墨汁灌注、TTC染色、MRI成像结果对该模型进行评价。结果大鼠术后状态良好,实验组大鼠观察4周后死亡率低为6 .25 %。大鼠神经功能评分均为1分,墨汁灌注及TTC染色观察梗死范围局限于皮层,4周后MRI成像测量梗死体积稳定,平均为83 .52 mm3。结论该模型对大鼠创伤小,梗死灶的位置和体积恒定,长期存活率高,为脑梗死后神经干细胞移植的研究提供了一种理想的动物模型。

Modeling postpartum depression in rats： theoretic and methodological issues

The postpartum period is when a host of changes occur at molecular, cellular, physiological and behavioral levels to prepare female humans for the challenge of maternity. Alteration or prevention of these normal adaptions is thought to contribute to disruptions of emotion regulation, motivation and cognitive abilities that underlie postpartum mental disorders, such as postpartum depression. Despite the high incidence of this disorder, and the detrimental consequences for both mother and child, its etiology and related neurobiological mechanisms remain poorly understood, partially due to the lack of appropriate animal models. In recent decades, there have been a number of attempts to model postpartum depression disorder in rats. In the present review, we first describe clinical symptoms of postpartum depression and discuss known risk factors, including both genetic and environmental factors. Thereafter, we discuss various rat models that have been developed to capture various aspects of this disorder and knowledge gained from such attempts. In doing so, we focus on the theories behind each attempt and the methods used to achieve their goals. Finally, we point out several understudied areas in this field and make suggestions for future directions.

Correlation between TIMP-1 expression and liver fibrosis in two rat liver fibrosis models

AIM： To evaluate serum TIMP-1 level and the correlation between TIMP-1 expression and liver fibrosis in immuneinduced and CCL4-induced liver fibrosis models in rats. METHODS： Immune-induced and CCL4-induced liver fibrosis models were established by dexamethasone （0.01 mg） and CCL4 respectively. Serum TIMP-1 level was detected with ELISA, while histopathological grade of liver biopsy was evaluated. Spearman rankcorrelation test was used to analyse the difference of the correlation between the TIMP-1 expression and hepatic fibrosis in the two fibrosis models. Furthermore,in situ hybridization was used to determine the expression difference of TIMP-1 mRNA in the two models. RESULTS： Positive correlation existed between serum TIMP-1 level of immune induced group and the histopathological stages of fibrosis liver of corresponding rats （Spearman rank-correlation test, rs = 0.812, P 0.05）, and the positive in situ hybridization signal of TIMP-1 mRNA was strong. In CCL4-induced liver fibrosis model, the correlation between the serum TIMP-1 level and the severity of hepatic fibrosis was not statistically significant（Spearman rank-correlation test, rs = 0.229, P 〉 0.05）. And compared with immune-induced model, the positivein situ hybridization signal of TIMP-1 mRNA was weaker, while the expression variation was higher in hepatic fibrosis of the same severity. CONCLUSION： The correlations between TIMP-1 expression and liver fibrosis in two rat liver fibrosis models are different. In immune-induced model, serum TIMP-1 level could reflect the severity of liver fibrosis, while in CCL4-induced model, the correlation between the serum TIMP-1 level and the severity of hepatic fibrosis was not statistically significant.

Establishing an experimental rat model of photo-dynamically-induced retinal vein occlusion using erythrosin B

AIM:To develop a reliable,reproducible rat model of retinal vein occlusion(RVO)with a novel photosensitizer(erythrosin B)and study the cellular responses in the retina.METHODS:Central and branch RVOs were created in adult male rats via photochemically-induced ischemia.Retinal changes were monitored via color fundus photography and fluorescein angiography at 1 and 3h,and 1,4,7,14,and 21d after irradiation.Tissue slices were evaluated histopathologically.Retinal ganglion cell survival at different times after RVO induction was quantified by nuclear density count.Retinal thickness was also observed.RESULTS:For all rats in both the central and branch RVO groups,blood flow ceased immediately after laser irradiation and retinal edema was evident at one hour.The retinal detachment rate was 100%at 3h and developed into bullous retinal detachment within 24h.Retinal hemorrhages were not observed until 24h.Clearance of the occluded veins at 7d was observed by fluorescein angiography.Disease manifestation in the central RVO eyes was more severe than in the branch RVO group.A remarkable reduction in the ganglion cell count and retinal thickness was observed in the central RVO group by 21d,whereas moderate changes occurred in the branch RVO group.CONCLUSION:Rat RVO created by photochemicallyinduced ischemia using erythrosin B is a reproducible and reliable animal model for mimicking the key features of human RVO.However,considering the 100%rate ofretinal detachment,this animal model is more suitable for studying RVO with chronic retinal detachment.

Potential rat model of anxiety-like gastric hypersensitivity induced by sequential stress

AIM To establish a rat model of anxiety-like gastric hyper-sensitivity(GHS) of functional dyspepsia(FD) induced by novel sequential stress.METHODS Animal pups were divided into two groups from postnatal day 2: controls and the sequential-stress-treated. The sequential-stress-treated group received maternal separation and acute gastric irritation early in life and restraint stress in adulthood; controls were reared undisturbed with their mothers. Rats in both groups were followed to adulthood(8 wk) at which point the anxietylike behaviors and visceromotor responses to gastric distention(20-100 mm Hg) and gastric emptying were tested. Meanwhile, alterations in several anxiety-related brain-stomach modulators including 5-hydroxytryptamine(5-HT), γ-aminobutyric acid(GABA), brain-derived neurotrophic factor(BDNF) and nesfatin-1 in the rat hippocampus, plasma and gastric fundus and the 5-HT1 A receptor(5-HT1 AR) in the hippocampal CA1 subfield and the mucosa of the gastric fundus were examined.RESULTS Sequential-stress-treated rats simultaneously demonstrated anxiety-like behaviors and GHS in dose-dependent manner compared with the control group. Although rats in both groups consumed similar amount of solid food, the rate of gastric emptying was lower in the sequentialstress-treated rats than in the control group. Sequential stress significantly decreased the levels of 5-HT(51.91 ± 1.88 vs 104.21 ± 2.88, P < 0.01), GABA(2.38 ± 0.16 vs 5.01 ± 0.13, P < 0.01) and BDNF(304.40 ± 10.16 vs 698.17 ± 27.91, P < 0.01) in the hippocampus but increased the content of nesfatin-1(1961.38 ± 56.89 vs 1007.50 ± 33.05, P < 0.01) in the same site; significantly decreased the levels of 5-HT(47.82 ± 2.29 vs 89.45 ± 2.61, P < 0.01) and BDNF(257.05 ± 12.89 vs 536.71 ± 20.73, P < 0.01) in the plasma but increased the content of nesfatin-1 in it(1391.75 ± 42.77 vs 737.88 ± 33.15, P < 0.01); significantly decreased the levels of 5-HT(41.15 ± 1.81 vs 89.17 ± 2.31, P < 0.01) and BDNF(226.49 ± 12.10 vs 551.36 ± 16.47, P < 0.01) in the gastric fundus but increased the content of nesfatin-1 in the same site(1534.75 ± 38.52 vs 819.63 ± 38.04, P < 0.01). The expressions of 5-HT1 AR in the hippocampal CA1 subfield and the mucosa of the gastric fundus were down-regulated measured by IHC(Optical Density value: Hippocampus 15253.50 ± 760.35 vs 21149.75 ± 834.13; gastric fundus 15865.25 ± 521.24 vs 23865.75 ± 1868.60; P < 0.05, respectively) and WB(0.38 ± 0.01 vs 0.57 ± 0.03, P < 0.01)(n = 8 in each group). CONCLUSION Sequential stress could induce a potential rat model of anxiety-like GHS of FD, which could be used to research the mechanisms of this intractable disease.

Key factors in developing the trinitrobenzene sulfonic acid-induced post-inflammatory irritable bowel syndrome model in rats

AIM:To investigate the key factors in developing the trinitrobenzene sulfonic acid(TNBS)-induced postinflammatory irritable bowel syndrome(PI-IBS)model in rats. METHODS:TNBS was administered to rats at the following conditions:(1)with different doses(20,10,5 mg/0.8 mL per rat);(2)with same dose in different concentrations(20 mg/rat,25,50 mg/mL);(3)in different ethanol percentage(25%,50%);and(4)at depth either 4 cm or 8 cm from anus.At 5 d and 4 wk after TNBS administration,inflammation severity and inflammation resolution were evaluated.At 4 and 8 wk after TNBS application,visceral hyperalgesia and enterochromaffin(EC)cell hyperplasia were assayed by abdominal withdrawal reflex test,silver staining and capillary electrophoresis. RESULTS:Our results showed that:(1)TNBS induced dose-dependent acute inflammation and inflammation resolution.At 5 d post TNBS,the pathological score and myeloperoxidase(MPO)activity in all TNBS treated rats were significantly elevated compared to that of the control(9.48±1.86,8.18±0.67,5.78± 0.77 vs 0,and 3.55±1.11,1.80±0.82,0.97±0.08 unit/mg vs 0.14±0.01 unit/mg,P<0.05).At 4 wk post TNBS,the pathological score in high and median dose TNBS-treated rats were still significantly higher than that of the control(1.52±0.38 and 0.80±0.35 vs 0,P<0.05);(2)Intracolonic TNBS administration position affected the persistence of visceral hyperalgesia.At 4 wk post TNBS,abdominal withdrawal reflex (AWR)threshold pressure in all TNBS-treated groups were decreased compared to that of the control(21.52 ±1.73 and 27.10±1.94 mmHg vs 34.44±1.89 mmHg,P<0.05).At 8 wk post TNBS,AWR threshold pressure in 8 cm administration group was still significantly decreased(23.33±1.33 mmHg vs 36.79±2.29 mmHg,P<0.05);(3)Ethanol percentage affected the TNBS-induced inflammation severity and visceral hyperalgesia.In TNBS-25%ethanol-treated group,the pathological score and MPO activity were significantly lowered compared to that of the TNBS-50%ethanoltreated group,while AWR threshold pressure were significantly elevated(36.33±0.61 mmHg vs 23.33±1.33 mmHg,P<0.05);and(4)TNBS(5 mg/0.8 mL per rat, in 50%ethanol,8 cm from anus)-treated rats recovered completely from the inflammation with acquired visceral hyperalgesia and EC cell hyperplasia at 4 wk after TNBS administration.CONCLUSION:TNBS dosage,concentration,intraco-lonic administration position,and ethanol percentage play important roles in developing visceral hyperalgesia and EC cell hyperplasia of TNBS-induced PI-IBS rats.

Detection and differentiation of early hepatocellular carcinoma from cirrhosis using CT perfusion in a rat liver model

BACKGROUND： Functional imaging such as CT perfusion can detect morphological and hemodynamic changes in he- patocellular carcinoma （HCC）. Pre-carcinoma and early HCC nodules are difficult to differentiate by observing only their hemodynamics changes. The present study aimed to investi- gate hemodynamic parameters and evaluate their differential diagnostic cut-off between pre-carcinoma and early HCC nodules using CT perfusion and receiver operating characteristic （ROC） curves. METHODS： Male Wistar rats were randomly divided into control （n=20） and experimental （n=70） groups. Diethylnitrosamine （DEN） was used to induce pre-carcinoma and early HCC nodules in the experimental group. Perfusion scanning was carried out on all survival rats discontinuously from 8 to 16 weeks. Hepatic portal perfusion （HPP）, hepatic arterial fraction （HAF）, hepatic arterial perfusion （HAP）, hepatic blood volume （HBV）, hepatic blood flow （HBF）, mean transit time （MTT） and permeability of capillary vessel surface （PS） data were provided by mathematical deconvolution model. The perfusion parameters were compared among the three groups of rats （control, pre-carcinoma and early HCC groups） using the Kruskal-Wallis test and analyzed with ROC curves. Histological examination of the liver tissues with hematoxylin and eosin staining was performed after CT scan.RESULTS： For HPP, HAF, HBV, HBF and MTT, there were significant differences among the three groups （P〈0.05）. HAF had the highest areas under the ROC curves： 0.80 （control vs pre-carcinoma groups） and 0.95 （control vs early HCC groups） with corresponding optimal cut-offs of 0.37 and 0.42, respectively. The areas under the ROC curves for HPP was 0.79 （control vs pre-carcinoma groups） and 0.92 （control vs early HCC groups） with corresponding optimal cut-offs of 136.60 mL/min/100 mg and 108.47 mL/min/100 mg, respectively. CONCLUSIONS： CT perfusion combined with ROC curve analysis is a new diagnosis model for distinguishing between pre-carcinoma and early HCC nodules. HAF and HPP are the ideal reference indices.

A chronic ulcerative colitis model in rats

INTRODUCTIONIn recent years,there have been many reports aboutanimal model to investigate drugs for inflammatorybowel diseases (IBD).The experimental animalmodel often used is acetic acid-induced damage ofcolonic muscosa.In the present study,this animalmodel was investigated by administering variousconcentrations of TNBS.

Neuroprotective effect of systemic and/or intravitreal rosuvastatin administration in rat glaucoma model

AIM： To evaluate the neuroprotective effect of rosuvastatin, in a rat experimental glaucoma model. METHODS： Ocular hypertension was induced in right eyes of Long-Evans rats （n=30） by cauterization of three episcleral veins. Left eyes were defined as controls. Rats were divided into five groups： oral rosuvastatin, intravitreal rosuvastatin, oral ＋intravitreal rosuvastatin, intravitreal sham and glaucoma without intervention. Rats were sacrificed at day 14. Retinal ganglion cell （RGC） number was assessed by histopathological analysis. Terminal deoxynucleotidyl transferase-mediated dUTP-nick end-labeling （TUNEL） staining and the expression of glial fibrillary acidic protein （GFAP） in RGC layer was also examined. RESULTS： A significant intraocular pressure （lOP） elevation was seen （P=0.002）. Elevated lOP resulted in a significant decrease in number of RGCs in group 5 （70.33 ±8.2 cells/mm2） when compared with controls （92.50 ±13.72 cells/mm2; P=0.03）. The RGC number in group 1 （92.4±7.3 cells/mm2） was significantly higher than group 5 （ρ=0.03）. The numbers of RGC in groups 2, 3 （57.3±8.2 cells/mm2, 60.5±12.9 cells/mm2） were comparable with that of group 5 （ρ=0.18 and P=0.31）. The apoptosis rates with TUNEL staining were also parallel to RGC number. Animals with experimentally induced glaucoma showed an increase in retinal GFAP immunoreacUvity. CONCLUSION： Decrease in RGC loss and apoptosis suggest the neuroprotective potential of oral rosuvastatin treatment in a rat model of ocular hypertension. Howeverintravitreal rosuvastatin showed a contrary effect and further studies are required.

Alleviated mucosal and neuronal damage in a rat model of Crohn's disease

AIM:To establish a rat model suitable to investigate the repetitive relapsing inflammations(RRI)characteristic to Crohn’s disease.METHODS:Colitis was induced by 2,4,6-trinitrobenzenesulfonic acid(TNBS).RRI were mimicked by repeating administrations of TNBS.Tissue samples were taken from control,once,twice and three times treated rats from the inflamed and adjacent non-inflamed colonic segments at different timepoints during the acute intestinal inflammation.The means of the ulcerated area were measured to evaluate the macroscopic mu-cosal damage.The density of myenteric neurons was determined on whole mounts by Hu C/Hu D immunohistochemistry.Heme oxygenase-1(HO-1)expression was evaluated by molecular biological techniques.RESULTS:TNBS-treated rats displayed severe colitis,but the mortality was negligible,and an increase of body weight was characteristic throughout the experimental period.The widespread loss of myenteric neurons,and marked but transient HO-1 up-regulation were demonstrated after the first TNBS administration.After repeated doses the length of the recovery time and extent of the ulcerous colonic segments were markedly decreased,and the neuronal loss was on a smaller scale and was limited to the inflamed area.HO-1 m RNA level was notably greater than after a single dose and overexpression was sustained throughout the timepoints examined.Nevertheless,the HO-1protein up-regulation after the second TNBS treatment proved to be transient.Following the third treatment HO-1 protein expression could not be detected.CONCLUSION:Experimentally provoked RRI may exert a protective preconditioning effect against the mucosal and neuronal damage.The persistent up-regulation of HO-1 m RNA expression may correlate with this.

A modified rat model for hepatocellular carcinoma

BACKGROUND: Rat hepatocellular carcinoma ( HCC ) model which has a high analogy to clinical liver cancer is of great value in understanding the pathogenesis and evolution of liver cancer, in searching effective anti-cancer treat- ments ( drug, hepatectomy and liver transplantation ), and designing cancer prevention strategies. In this study we es- tablished a modified rat model of hepatocellular carcinoma to enhance rats' physique and surgical endurance. METHODS: Wistar rats were fed with diethylnitrosamine (DENA) by three methods for evaluation of general condi- tions for 130 days: Doppler ultrasonographic measurement, laparotomy and histopathological examination. RESULTS: No rat died in control group ( group A) and modified DENA-induction-HCC group ( group C), but 6 deaths in classical DENA-induction-HCC group (group B) (survival rate 80%). All survived rats in groups B and C de- veloped diffusive hepatocellular carcinoma and liver cirrho- sis. General appearance of rats in the group C was better than that in the group B. CONCLUSION: With good general conditions for surgery, the modified rat model for hepatocellular carcinoma has a high carcinogenic rate and a high survival rate.

A rat model of high altitude polycythemia rapidly established by hypobaric hypoxia exposure

Objective To investigate the effects of simple hypobaric hypoxia on parameters of hematology and blood rheology in order to establish a rat model of simulated high altitude polycythemia(HAPC) for the study of pathophysiologic mechanisms and medical prevention and treatment of HAPC.Methods Fortyeight male Wistar rats were randomly divided into three normal control groups and three hypoxia model groups.Normal control group rats were bred in normoxia conditions,and hypoxia group rats were subjected to hypoxic exposure for 8 hours per day at simulated 5 500 m high altitude in a hypobaric chamber.After hypoxic exposure for 2,4,12 weeks,one group of normal control and hypoxia model rats were killed and blood was collected,respectively.Then parameters of erythrocyte and blood rheology were examined.Results Mucous membrane of hypoxia model rats showed obviously cyanosis after 2 weeks hypoxic exposure.Hemoglobin concentration of hypoxia model rats were beyond 210 g/L after 2 weeks,4 weeks and 12 weeks hypoxia exposure and significantly increased than that of normal control rats respectively.Besides,RBC counts,hematocrit,whole blood viscosity,erythrocyte aggregation index of hypoxia model rats were all notably higher than those of normal control rats respectively.Conclusion A rat model of high altitude polycythemia can be rapidly established by hypobaric hypoxia exposure at simulated 5 500 m high altitude for 8 hours daily.

A Comparative Study on Several Models of Experimental Renal Calcium Oxalate Stones Formation in Rats

In order to compare the effects of several experimental renal calcium oxalate stones formation models in rats and to find a simple and convenient model with significant effect of calcium oxalate crystals deposition in the kidney, several rat models of renal calcium oxalate stones formation were induced by some crystal-inducing drugs （CID） including ethylene glycol （EG）, ammonium chloride （AC）, vitamin D3 [ 1 α（OH）VitD3, alfacalcidol], calcium gluconate, ammonium oxalate, gentamicin sulfate, L-hydroxyproline. The rats were fed with drugs given singly or unitedly. At the end of experiment, 24-h urines were collected and the serum creatinine （Cr）, blood urea nitrogen （BUN）, the extents of calcium oxalate crystal deposition in the renal tissue, urinary calcium and oxalate excretion were measured. The serum Cr levels in the stone-forming groups were significantly higher than those in the control group except for the group EG＋L-hydroxyproline, group calcium gluconate and group oxalate. Blood BUN concentration was significantly higher in rats fed with CID than that in control group except for group EG＋L-hydroxyproline and group ammonium oxalate plus calcium gluconate. In the group of rats administered with EG plus Vitamin D3, the deposition of calcium oxalate crystal in the renal tissue and urinary calcium excretion were significantly greater than other model groups. The effect of the model induced by EG plus AC was similar to that in the group induced by EG plus Vitamin D3. EG plus Vitamin D3 or EG plus AC could stably and significantly induced the rat model of renal calcium oxalate stones formation .