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.

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.

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.

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.

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 .

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.

Establishment of an Infected Necrotizing Pancreatitis Model by Retrograde Pancreatic Duct Injection of Sodium Taurocholate and E. coli in Rats

A stable and reliable infected necrotizing pancreatitis （INP） model in rats was established in order to study the pathophysiological mechanism and pathological development role of INP and explore the new therapeutic methods for the diseases. Forty-six SD rats were randomly divided into 5 groups. The animals in group A received the injection of 5% sodium taurocholate into the pancreatic duct and those in group B underwent that of E. coli into the pancreatic duct. The rats in groups C, D and E were subjected to the injection of 5% sodium taurocholate in combination with different concentrations of E. coli （10^3, 10^4, 10^5/mE, respectively） into the pancreatic duct. The dose of injection was 0.1 mL/100 g and the velocity of injection was 0.2 mL/min in all the 5 groups. Eight h after the injection, the survival rate of animals was recorded and the surviving rats were killed to determine the serum content of amylase and perform pathological examination and germ cultivation of the pancreatic tissue. The results showed that acute necrotizing pancreatitis model was induced by injection of 5% sodium taurocholate into the pancreatic duct. The positive rate of germ cultivation in group A was 12.5%. The acute necrotizing pancreatitis model was not induced by injection of E. coli into the pancreatic duct and the positive rate of germ cultivation in group B was 0. The INP model was established in groups C to E. The positive rate of germ cultivation was 60%, 100% and 100% and 8-h survival-rate 100%, 100% and 70% in groups C, D and E, respectively. It was concluded that a stable and reliable model of INP was established by injection of 5% sodium taurocholate in combination with 10^4/mL E. coli into the pancreatic duct with a dose of 0.1 mL/100 g and a velocity of 0.2 mL/min. The pathogenesis of INP might be that the hemorrhage and necrosis of pancreatic tissue induced by sodium taurocholate results in weakness of pancreatic tissue in fighting against the germs. Meanwhile, the necrotic pancreatic tissue provides a good proliferative environment for the germs.

Effects of C2 hemisection on respiratory and cardiovascular functions in rats

High ce rvical spinal co rd injuries induce permanent neuromotor and autonomic deficits.These injuries impact both central respiratory and cardiovascular functions through modulation of the sympathetic nervous system.So far,cardiovascular studies have focused on models of complete contusion or transection at the lower cervical and thoracic levels and diaphragm activity evaluations using invasive methods.The present study aimed to evaluate the impact of C2 hemisection on different parameters representing vital functions(i.e.,respiratory function,cardiovascular,and renal filtration parameters)at the moment of injury and 7 days post-injury in rats.No ventilatory parameters evaluated by plethys mography were impacted during quiet breathing after 7 days post-injury,whereas permanent diaphragm hemiplegia was observed by ultrasound and confirmed by diaphragmatic electromyography in anesthetized rats.Interestingly,the mean arterial pressure was reduced immediately after C2 hemisection,with complete compensation at 7 days post-injury.Renal filtration was unaffected at 7 days post-injury;however,remnant systolic dysfunction chara cterized by a reduced left ventricular ejection fraction persisted at 7 days post-injury.Taken together,these results demonstrated that following C2 hemisection,diaphragm activity and systolic function are impa cted up to 7 days post-injury,whereas the respiratory and cardiovascular systems display vast ada ptation to maintain ventilatory parameters and blood pressure homeostasis,with the latter likely sustained by the remaining descending sympathetic inputs spared by the initial injury.A better broad characterization of the physiopathology of high cervical spinal cord injuries covering a longer time period post-injury could be beneficial for understanding evaluations of putative therapeutics to further increase cardiorespiratory recovery.

Mechanism of AiTongXiao granule in the treatment of hepatocellular carcinoma based on network pharmacology and rat transplanted liver cancer model

Objective:To investigate the mechanism of action and material basis of AiTongXiao granule in the treatment of hepatocellular carcinoma(HCC)based on network pharmacology and transplanted liver cancer rat model.Methods:TCMSP database was used to screen out effective components and its corresponding potential pharmaceutical targets,and databases including Gene Cards,OMIM,Drugbank and TTD were further used to collect HCC-related drug targets.The intersecting targets were obtained by mapping the drug and disease targets.The component-targets network was constructed and visualized by Cytoscape 3.8.2 software.Protein-protein interaction(PPI)network was built by STRING online platform,and the topological relationship and core targets was analyzed and screened by using CytoNCA software.In addition,Metascape database was used to perform gene ontology(GO)enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis of the core targets.At last,rat liver transplanted liver cancer model was established by using Walker-256 cell line and treated by AiTongXiao granule for 15 days.Western blot was used to further compare the expression levels of AKT,pAKT,p53,p-p53,ERK1/2 and ERK1/2 in the tumor between treatment group and the control group.Results:257 active components were obtained from AiTongXiao granule,corresponding to 294 drug targets.Meanwhile,233 of the 7993 HCC disease targets were screened out between AiTongXiao granule drug and HCC disease targets.11 core targets including AKT1,IL6,TP53,MAPK3,TNF,JUN,CASP3,MAPK1,MYC,PTGS2,MMP9 were further obtained by median screening.GO and KEGG analysis results showed that these core targets enriched to HBV,TNF and cancer related pathways.The rat transplanted liver cancer model results indicated significant down regulation for AKT,p-AKT,pERK1/2,and significant up regulation of p-p53 after AiTongXiao granule treatment(P<0.05).Conclusion:AiTongXiao granule could act to multiple cancer related pathways,and AKT,p53 and ERK1/2 were validated to be regulated by ATXF in rat model.The mechanism may be through the regulation of the above signaling pathways to exert anti-liver cancer effect.

Construction of a model of autologous blood intracerebral hemorrhage in rats with a double injection and double needle withdrawal and the characteristics of the operative technique

BACKGROUND ： Experimental animal models of intracerebral hemorrhag （ICH） are greatly needed, so the process of establishment should be ideal in hematoma formation and easy to operate. OBJECTIVE ： To construct model of ICH in rats with double injection of autologous blood taken from the cut tail cut and double withdrawal of the needle （shortened as two-step injection model）, and compare with those induced by single and double injections. DESIGN ： A randomized controlled tria SETTING： Department of Neurology, General Hospital of Military Area Command of Chinese PLA. MATERIALS： Thirty male Wistar rats of 10 to 12 months, weighing （400±25） g, provided by the Experimental Center of Medical Animals, General Hospital of Shenyang Military Area Command of Chinese PLA, were divided randomly into 3 groups with 10 rats in each group： two-step injection group, single injection group, double injection group. METHODS ： The experiment was carried out in the Department of Neurology, General Hospital of Military Area Command of Chinese PLA from March to June in 2004. Autologous blood ICH model in rats were established as follows： In the two-step injection group, 50 μL unclotted autologous blood was taken from the rat tail cut, then injected with microsyringe into the caudate nucleus, 10 μL injected at first, paused for 2 minutes, and then the rest 40 μL injected slowly and continuously within 2 minutes. After the injection, the needle was kept immovable for about 4 minutes, withdrawn 2.0 mm, again kept immovable for about 4 minute, and then removed wholly at a slow speed. In the single injection group, 50 μL unclotted tail blood was injected slowly and continuously all within 2 minutes and the needle was slowly removed;（4） In the double injection group, 10 mL blood was injected at first, paused for 2 minutes, the rest 40 μL injected evenly within 2 minutes, and then the needle was withdrawn slowly and uninterruptedly. Neurologic findings were scored in accordance with Longa＇s five-point scale （0-4 scores, the higher the score, the severer the neurological dysfunction）. The rats were killed to remove and sections were prepared, the morphological features of hematomas were grossly observed, the maximal diameter and size of hematomas in each slice were measured with the imaging analytical system, and the volume was calculated. Meanwhile, the conveniences of the techniques were compared. MAIN OUTCOME MEASURES： The morphological features and volume of hematomas, neurologic deficit score （NDS）, and the convenience of the techniques were compared. RESULTS： All the 30 rats were involved in the analysis of results without deletion. （1） Results of the morphological observation of volume of hematoma： In the two-step injection group, hematomas located in the right caudate nucleus area regularly in circular or analogously circular shape in each slice. The formation rate of hematomas in the single injection group and double injection group were lower than those in the two-step injection group [60% （6/10）, 80% （8/10）, 100% （10/10）, P〈 0.01, 0.05]. The volume of hematomas in the single injection group and double injection group were smaller than those in the two-step injection group [（28.5±14.8）, （33.4±7.4）, （41.6±3.9） mm3, P〈 0.01, 0.05]. （2） NDS results： The NDS scores in the single injection group and double injection group were smaller than that in the two-step injection group （0.90±0.83, 1.30±0.78, 1.90±0.57, P〈 0.05）. （3）Comparison of the convenience of the techniques： The double injection method allowed generating reproducible hematomas in rats with shortcomings that it needed autologous arterial blood from femoral artery, and precision instruments such as microinfusion pump. The two-step injection injected fresh unclotting blood taken directly from the tail cut with microsyringe into the rat brain, and it has the advantages of easy operation, no influence on the activity of thrombase, shorter duration for model establishment, and higher rate of hematoma formation, which could generate ideal and economical models of ICH. The two-step injection induced hemotoma regularly in circular or analogously circular shape in each slice, but those induced by single and double injections were mostly in strip or fusiform shapes and extending along the needle tracks or into the ventricle or subarachnoid space. CONCLUSION ： The autologous blood ICH model induced by the two-step injection method is a reproducible and reliable one in regular shape, which is better than those induced by double and single injections.

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.

Efficacy of Chinese medicine Yi-gan-kang granule in prophylaxis and treatment of liver fibrosis in rats

AIM:To investigate the efficacy of a Chinese medicine, Yi-gan-kang granule (granules for benefiting the liver), in prophylaxis and treatment of liver fibrosis in rats and its possible mechanism. METHODS: One hundred and forty Sprague-Dawley rats were randomly divided into seven groups (20 each): group 1, blank control group without any interference during the study; group 2, CCI4-induced liver fibrosis group; group 3, pig serum-induced liver fibrosis group; group 4, prophylaxis group of CCl4-induced liver fibrosis by Yi-gan-kang; group 5, prophylaxis group of pig serum-induced liver fibrosis by Yi-gan-kang; group 6, treatment group of CCI4-induced liver fibrosis by Yi-gan-kang; group 7, treatment group of CCI4-induced liver fibrosis by Yi-gan-kang. At wk 6,10,14 and 20 (baseline for CCl4., or big serum induction), five rats in each group were anesthetized and their livers were removed for pathological studies including immunohistochemical studies for α-SMA, type I collagen and In situ hybridization of tissue inhibitor of metalloproteinase-1 (TTMP-1) mRNA of hepatic stellate cells (HSCs). Anti-lipid peroxidation in isolated mitochondria and 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) colorimetric assay for proliferation and terminal deoxynucleotidyl transferase-medicated dUTP-biotin nick end-labeling (TUNEL), flow cytometry and electron microscopy for apoptosis in isolated HSCs were also studied. RESULTS: The mean number of pseudolobuli at wk 10, 14 and 20 in the prophylaxis group was significantly less than that in the control group (P<0.05 or 0.01). The effect of prophylaxis at wk 14 in CCI4 rats and at wk 10 in pig serum-induced rats was much better than that of treatment group (P<0.01). The thickness (in μm) of fibers both in pig serum-induced prophylaxis and in treatment groups at wk 14 and. 20 was significantly less than that in control group (P<0.05). The number of fibers both in prophylaxis and in treatment groups from wk 10 or 14 to 20 was significantly less than that in control group (P<0.05 or P<0.01). The tissue HSC positive rates of type I collagen, α-SMA and TIMP-1 mRNA, which represented the active phenotype of HSCs in tissues, remained very high from wk 6 to the end of model making in control group. While in prophylaxis group, they were at a relatively low level. In treatment group, there was a gradual decreasing trend. Time- and dose-dependent effects of anti-lipid peroxidation on isolated mitochondria, cell proliferation and apoptosis in cultured HSCs were also observed during the study. CONCLUSION: Yi-gan-kang can effectively inhibit or inverse the course of liver fibrogenesis in CCI4- and pig serum-induced rat models.

Effects of Haobie Yangyin Ruanjian Decoction on hepatic fibrosis induced by carbon tetrachloride in rats

AIM:To explore the anti-fibrotic effect of Haobie Yangyin Ruanjian Decoction(HYRD)on CCl4-induced hepatic fibrosis in rats and its modulation on the transforming growth factor(TGF)β-Smad signaling pathway.METHODS:Fifty-six healthy Wistar rats were randomly divided into five groups:normal control group(n=6),CCl4-induced hepatic fibrosis group(n=14) and three treatment groups(the treated rats received HYRD via oral administration at daily dosages of 8.2,2.5 and 0.82 g/kg,respectively)of HYRD(n=12,respectively).Experimental hepatic fibrosis was induced by subcutaneous injection of carbon tetrachloride solution(CCl4 dissolved in peanut oil,4:6,V/V)with 0.5 mL/100 g body weight for the first time,and then 0.3 mL/100 g body weight twice a week for 8 wk.In the former 2 wk,rats were raised by feedstuffⅠ(80% corn meal,20%lard,0.5%cholesterol).After 2 wk,they were raised by feedstuffⅡ(corn meal and 0.5% cholesterol).Except for the control group,30%alcohol solution was given orally to each rat every other day from the beginning,1 mL for each rat.Liver function parameters and hepatic hydroxyproline content were detected by chromatometry.Serum levels of hyaluronic acid(HA),typeⅣcollagen(CIV),typeⅢprecollagen(PCⅢ)and laminin(LN)were assayed with radioimmunoassay.Deposition of collagen was observed with hematoxylin-eosin staining and collagen staining.Gene expression of TGFβ1 and Smad3 were detected with real-time reverse transcriptase-polymerase chain reaction and Western blotting,respectively.RESULTS:The serum levels of alanine transaminase and aspartate transaminase were increased in the model group compared with the control group(P<0.01),and they were decreased in the three treatment groups compared with the model group.The serum levels of total protein and albumin were decreased in the model group and increased in the three treatment groups.The hepatic hydroxyproline content and serum levels of PCⅢ,HA,LN and CIV were markedly increased in the model group compared with the control group,and decreased in the treatment groups.The gene expression of TGFβ1 and Smad3 was enhanced in the model group compared with the control group,and HYRD could down regulate their expression.CONCLUSION:HYRD can inhibit hepatic fibrosis induced by CCl4 in rats,which is probably associated with its down-regulation on fibrogenic signal transduction of TGFβ-Smad pathway.

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.

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.

Neuroprotective effect of baicalin on focal cerebral ischemia in rats

Baicalin, a flavonoid compound from the root of the herb Scutellaria baicalensis Georgi, has been widely used to treat patients with inflammatory disease. The aim of this study was to assess the efficacy of baicalin in a rat model of focal cerebral ischemia. Adult male Sprague-Dawley rat models of cerebral artery occlusion were established and then randomly and equally divided into three groups： ischemia（cerebral ischemia and reperfusion）, valproic acid（cerebral ischemia and reperfusion ＋ three intraperitoneal injections of valproic acid; positive control）, and baicalin（cerebral ischemia and reperfusion ＋ intraperitoneal injection of baicalin for 21 days）. Neurological deficits were assessed using the postural reflex test and forelimb placing test at 3, 7, 14, and 21 days after ischemia. Rat cerebral infarct volume was measured using 2,3,5-triphenyltetrazolium chloride（TTC） staining method. Pathological change of ischemic brain tissue was assessed using hematoxylin-eosin staining. In the baicalin group, rat neurological function was obviously improved, cerebral infarct volume was obviously reduced, and the pathological impairment of ischemic brain tissue was obviously alleviated compared to the ischemia group. Cerebral infarct volume was similar in the valproic acid and baicalin groups. These findings suggest that baicalin has a neuroprotective effect on cerebral ischemia.

Antifi brotic effects of green tea on in vitro and in vivo models of liver fibrosis

AIM: To examine the protective effect of green tea extract (GT) on hepatic fi brosis in vitro and in vivo in dimethylnitrosamine (DMN)-induced rats.METHODS: HSC-T6, a rat hepatic stellate cell line, was used as an in vitro assay system. Cell proliferation, collagen content, and type 1 collagen expression were examined in activated HSC-T6 cells. Collagen was determined by estimating the hydroxyproline content. In rats with DMN-induced hepatic fi brosis, serum aspartate aminotransferase and alanine aminotransferase concentrations, liver hydroxyproline and lipid peroxides were determined. Pathologic changes were examined by hematoxylin & eosin staining.RESULTS: GT administration prevented the development of hepatic fibrosis in the rat model of DMN-induced liver fi brosis. These results were confi rmed both by liver histology and by quantitative measurement of hepatic hydroxyproline content, a marker of liver collagen deposition. Accordingly, inhibition of proliferation, reduced collagen deposition, and type 1 collagen expression were observed in activated HSC-T6 cells following GT treatment. These results imply that GT reduced the proliferation of activated HSC and down regulated the collagen content and expression of collagen type 1, thereby ameliorating hepatic fibrosis.CONCLUSION: This study demonstrates that greentea administration can effectively improve liver fibrosis caused by DMN, and may be used as a therapeutic option and preventive measure against hepatic f ibrosis.

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.