Preparation of a rat model of neonatal hypoxic-ischemic encephalopathy / 中国比较医学杂志

Objective To simulate the process of hypoxic?ischemic brain injury at high altitude in a simulated cabin with plateau low pressure environment, and to prepare a rat model of cerebral injuries at different high altitudes. Method Thirty?two 0?day?old neonatal SD rats were divided into four groups, namely group A ( control group) and three test groups:group B (2000 m group), group C (4000 m group), and group D (6000 m group). The rats of control group were reared in a barrier environment. The rats of test groups were placed in a simulated cabin with plateau low pressure environment, and to prepare neonatal cerebral ischemia?hypoxia model by sport activities. The sport movements were carried out in the cabin in a swimming groove 60 min/d, and not less than 20 hours a day at high altitude low pressure environment. Zea Longa 5 point scale standard was used to determine the behavioral scores at the 3 th 7 th 11 th 15 th days, and samples were collected on the 15th day to observe red blood cell morphology using HE and 2, 3, 5?triphenyltetrazolium chloride ( TTC ) staining and ultrastructure by scanning electron microscopy. Result ( 1 ) The neurological scores of the test groups A, B, C were significantly different from that of the control group (P<0?05), and the scores of test group D and control group were very significantly different ( P <0?01 ) . ( 2 ) The histopathological examination using HE staining showed inflammatory cell infiltration in all rats of the test groups, and the extent of inflammatory cell infiltration was positively correlated with the increase of altitude. ( 3 ) The histopathology with TTC staining revealed prominent ischemia in the cerebral cortex of rats in the plateau hypoxic environment. ( 4 ) Scanning electron microscopy showed that the rat erythrocytes were cap?like in the group B, irregular in the group C, and zigzag shape in the group D. Conclusions In this study, a rat model of neonatal hypoxic?ischemic encephalopathy ( HIE) is successfully established by hypoxic cabin combined with sport activity. This model is stable, reliable, more closely mimicking the pathogenesis and clinical manifestation of neonatal HIE than models prepared with other methods, therefore, may be used in related research.

Three New Records of Mortierella Species Isolated from Crop Field Soil in Korea

Three new fungal species of the genus Mortierella, Mortierella zychae, Mortierella ambigua, and Mortierella indohii, have been reported in Korea. The fungi were encountered during a study on the fungal community of soil samples collected from different locations in Korea. The species were identified based on molecular and morphological analyses. This study presents detailed descriptions of the morphological observations and molecular phylogenetic analysis of these three fungi. All three species were found to be sensitive to triphenyltetrazolium chloride staining. M. zychae demonstrated the highest intensity of mycelial staining, indicating that this species has the highest potential to produce arachidonic acid of the three species. The staining results indicated that the newly recorded species could potentially be useful for arachidonic acid production.

A forced running wheel system with a microcontroller that provides high-intensity exercise training in an animal ischemic stroke model

We developed a forced non-electric-shock running wheel (FNESRW) system that provides rats with high-intensity exercise training using automatic exercise training patterns that are controlled by a microcontroller. The proposed system successfully makes a breakthrough in the traditional motorized running wheel to allow rats to perform high-intensity training and to enable comparisons with the treadmill at the same exercise intensity without any electric shock. A polyvinyl chloride runway with a rough rubber surface was coated on the periphery of the wheel so as to permit automatic acceleration training, and which allowed the rats to run consistently at high speeds (30 m/min for 1 h). An animal ischemic stroke model was used to validate the proposed system. FNESRW, treadmill, control, and sham groups were studied. The FNESRW and treadmill groups underwent 3 weeks of endurance running training. After 3 weeks, the experiments of middle cerebral artery occlusion, the modified neurological severity score (mNSS), an inclined plane test, and triphenyltetrazolium chloride were performed to evaluate the effectiveness of the proposed platform. The proposed platform showed that enhancement of motor function, mNSS, and infarct volumes was significantly stronger in the FNESRW group than the control group (P<0.05) and similar to the treadmill group. The experimental data demonstrated that the proposed platform can be applied to test the benefit of exercise-preconditioning-induced neuroprotection using the animal stroke model. Additional advantages of the FNESRW system include stand-alone capability, independence of subjective human adjustment, and ease of use.

An optimized modification of TTC staining method for better detection of myocardial ischemia-reperfusion injury in rats / 中国实验动物学报

Objective To evaluate an improved modification of TTC staining method for measuring myocardial in-farct size after ischemia-reperfusion in rats.Methods Twenty healthy SPF male 8-week-old SD rats were randomly divided into two groups:Group A with conventional TTC staining, and group B with the modified TTC staining method for measuring myocardial ischemia-reperfusion injury.The infarct size was caculated and the serum cTnI levels were determined.Results The infarcted myocardium was well detected in both groups A and B.There were no significant differences in the myocardial infarct sizes measured in the groups A and B (48.69 ±5.37 %vs.47.41 ±3.28%, P>0.05).There were no significant differences in the serum cTnI levels assayed in the groups A and B (4.51 ±0.88 ng/mL vs.4.70 ±0.71 ng/mL, P>0.05). But compared with the group A, the color contrast of stained myocardial slice and the distinguishing infarction area and non-infarction area were much clearer in the group B.Conclusions Our modified TTC staining technique using in vivo staining is an economic, convenient, fast and efficient method, being easy to control, time-saving and inexpensive, and enhances the staining effect in evaluating the size of myocardial ischemia/reperfusion injury more accurately.

Evaluation of a Colorimetric Broth Microdilution Method for Antimicrobial Susceptibility Testing Using 2,3,5-Triphenyltetrazolium Chloride / 대한임상미생물학회지

BACKGROUND: The broth microdilution susceptibility testing method is considered a standard for determining minimum inhibitory concentrations, and the addition of the redox indicator 2,3,5-triphenyltetrazolium chloride (TTC) to the broth microdilution method simplifies and increases its objectivity. The current study evaluated the usefulness of a TTC-modified broth microdilution method for antimicrobial susceptibility test of frequently encountered clinical isolates. METHODS: The minimal inhibitory concentrations (MICs) of 10 antimicrobials for 111 clinical isolates of four bacterial species, Staphylococcus aureus, Escherichia coli, Enterobacter cloacae, and Acinetobacter baumannii, were investigated by a modification of the Clinical and Laboratory Standards Institute (CLSI)-recommended broth microdilution method with the addition of 2,3,5-triphenyltetrazolium chloride (TTC). The inhibitory effects of TTC against 192 strains of 22 bacterial species isolated from clinical specimens were also evaluated. RESULTS: The number of colonies of all 192 strains of 22 bacterial species grown on TTC-containing Mueller-Hinton agar did not differ from those grown on Mueller-Hinton agar only. The MICs with TTC were within 2 dilutions of those obtained by the CLSI method in 569 (97.6%) of 583 organism-antimicrobial agent combinations. CONCLUSIONS: The colorimetric MIC method using TTC may be a useful surrogate of antimicrobial susceptibility testing for most of the frequently isolated bacteria.

Susceptibility Test of Methicillin-resistant Staphylococcus aureus against Vancomycin Using Triphenyltetrazolium Chloride / 감염

BACKGROUND: Abuse and inappropriate use of antibiotics have resulted in emergence of antibiotic-resistant microorganisms, increased nosocomial infections and urgency of developing new drugs effectively coping the resistant microorganisms. Recently infections caused by methicillin-resistant Staphylococcus aureus (MRSA) strains with reduced susceptibility to vancomycin (MIC=8 microgram/mL) have been reported in both Japan and the United States. Micro-broth dilution method and population analysis method revealed that those strains in Japan were heterogeneously resistant to vancomycin. The aim of this study was to determine the susceptibility and minimum inhibitory concentrations (MIC) of 99 MRSA strains to vancomycin by modified microplate-broth dilution method with triphenyltetrazolium chloride. METHODS: Susceptibility and minimum inhibitory concentration of stains of MRSA collected to vancomycin were determined by micro-plate broth dilution method with triphenyltetrazolium chloride solution, one of the growth indicator of microorganisms. RESULTS: MICs of 99 strains of MRSA isolated from otorrhea, pus, blood, respiratory tract, tissue fluid, and sputum to vancomycin were determined with microplate-broth dilution method with triphenyltetrazolium chloride. All the 99 strains showed MIC under 8 microgram/mL, there was no strain with reduced susceptibility to vancomycin. Microplate-broth dilution method with triphenyltetrazolium chloride was simple, easy to interpret the results. CONCLUSION: All the strains of MRSA tested for the susceptibility to vancomycin were susceptible to vancomycin. The microplate-broth dilution method with triphenyltetrazolium chloride was simple, easy to interpret the results and economic, and it is suggested that this method could be applied to the antimicrobial susceptibility tests of the other bacteria and fungi.

Measurement of Regional Tissue Water and Ionic Concentrations During the Evolution of Infraction and the Therapeutic Value of Nimodipine in Rat Middle Cerebral Artery Occlusion Model of Ischemia

The evolution of infarction in the rat middle cerebral artery(MCA) occlusion model was examined by atomic absorption spectrometric measurements of Na, K and Ca concentrations in brain tissue sample. At 2, 4, 6, 8, and 24 hours after MCA occlusion and sham occlusion the brain tissue samples were obtained. Tissue water concentrations were estimated from dry-wet weight measurement. The effects of nimodipine(2 microgram/kg/min for 10 min) administered intra-venously at 4 hours(Group A), 6 hours(Group B), and 9 hours(Group C) after MCA occlusion were investigated on both the size of infarction and tissue water, Na, K, Ca concentrations at 24 hours. The result were as follows : 1) Normal concentrations of water, Na, K, and Ca averaged 0.793+/-0.009ml, 54.06+/-4.18 micromole, 81.04+/-3.44 micromole, and 3.578+/-0.712 micromole/gm wet weight. At the infarct site by 24 hours, the changes of tissue water and ionic concentrations were conspicuously evident so that water increased by more than 10%(p<0.005), Na increased by more than 120%(p<0.005), K decreased by more than 75%(p<0.005), and Ca increased by more than 200%(p<0.005). 2) The remarkable shifts of Na, K, and Ca concentrations occurred at 4-6 hours so that 60-85% of the ionic shifts developed by 6 hours. This characteristics of chronological ionic changes correlated well with the findings of 2% TTC staining during the evolution of infarction. Water concentrations increased rapidly at 2-4 hours so that nearly 80% of water shift developed by 4 hours. 3) In group A(administered at 4 hours), nimodipine treatment significantly reduced both the ionic shifts at the infarct site and the size of infarction compared with non-treated rats(p<0.05). 4) In group B(administered at 6 hours), nimodipine treatment did not significantly reduce the ionic shifts but did reduce the size of infarction compared with non-treated rats(p<0.05). In group C(administered at 8 hours), nimodipine treatment significantly reduce neither the ionic shifts nor the size of infarction. In summary it was concluded that the progressive changes in tissue water and ionic concentrations developed at the infarct sites and the critical period of the changes was between 4 and 6 hours, and nimodipine treatment was effective when administered within 4 hours. The results suggested that measurement of tissue ionic concentrations could be used as an alternative method for assessing tissue damage and a reliable method to quantify the tissue damage. This method may be useful for determining the time window for therapeutic protocol, as well as for evaluating therapeutic effects.

The Evaluation and Quantification of Cerebral Infarction Following a Middle Cerebral Artery Occlusion in Rats

In order to find out the accuracy of the quantification of the infarction area by using triphenyltetrazolium chloride(TTC) staining and to evaluate the change of the infarction size according to the duration after the ischemic insult, in a series of 33 adult rats, a surgical occlusion of the middle cerebral artery(MCA) was carried out through a small subtemporal craniotomy. 11 animals at 6 hour, 12 animals at 24 hour and 10 animals at 48 hour following the surgical occlusion of the MCA, rats were sacrificed and brain slices were obtained and stained with TTC, and hematoxyline and eosin(H & E). The size of the infraction area stained by each method was quantified by a computer image analysis system. The average percent of the infarction size(+/-standard error) was larger in the 24 and 48 hour groups than that of the 6 hour group(determined by TTC:9.94+/-0.97 vs. 9.98+/-1.08 vs. 6.83+/-0.82%, respectively:6 hour vs. 24 & 48 hour groups;one-way ANOVA test p0.05), and linear regression analysis showed a significant correlation existed between the two methods in all groups. However, the degree of correlation was more prominent in the 24 and the 48 hour groups than 6 hour group(6 hour group:r=0.76, slope=0.78, y intercept=0.55;24 hour group:r=0.97, slope=1.03, y intercept=-0.78;48 hour group:r=0.98, slope=0.94, y intercept=0.42). From this study it is concluded that: 1) the evolution of the infarction size continues up to 24 hours following the arterial occlusion, and thereafter, the change of the infarction size is minimal in the rat. This data suggests that it is sufficient to evaluate the change of the infarction size up to 24 hours following the ischemic insult in the experimental study of ischemia in the rat. 2) the detection and the quantification of the cerebral infarction by using TTC staining is a reliable method after 24 hours following the ischemic insult. However, in the earlier period than 24 hours following the ischemic insult, staining with TTC coupled with histopathological H & E staining will add to the accuracy in the obtainin the quantity of the cerebral infarction in the rat.

Prolonged Ischemic Cerebral Infarct in the Rat after Middle Cerebral Artery Occlusion: Part 1:Evolution and Time Course of the Infarction

Although evolution and time course of ischemic brain infarct should be a matter of interest to investigators in the research of brain ischemia as well as traumatic brain injury, few papers have ever been reported. Authors observed quantitatively sequential changes of infarct size and regional cerebral blood flow(rCBF) to assess the evolution of focal ischemic brain infarct in the rat following left MCAO. Fifteen rats, weighting 250 g to 370 g, were used in this experiment. The experiment animals were divided into three groups: 6, 24 and 48 hours groups(HG) after MCAO. The rCBF of bilateral caudate nuclei was measured by hydrogen clearance methods. Areas of brain infarction were delineated by tripheny-tetrazolium chloride(TTC) at the preselected 8 coronal levels of forebrain. The areas of brain damage were drawn on scale diagrams(x4 actual size) of forebrain and measured by a plannimeter. In the experimental groups, just after MCAO, rCBF of the ipsilateral caudate nucleus was reduced to 29.4+/-6.5 to 24.5+/-7.9 ml/100 g/min from the basal value of around 117 to 121 ml/100 g/min and showed a tendency of getting more reduced to 19.4+/-7.6 ml/100 g/min by 48 hours. The rCBF of the contralateral caudate nucleus was maintained in the basal value throughout the experiment. Comparing the total amounts of ischemic damage of 48 HG to those of 5 and 24 HG, the infarction size was significantly increased in cerebral hemisphere as well as cerebral cortex and caudate nucleus(p<0.05). But there was not any significant difference between 6 and 24 HG. The experiment provides evidence for the evolution of focal ischemic brain infarct without any further change of decreased rCBF. The data suggest that it is desirable to observe the change of pathologic findings by not less then 48 hours following the arterial occlusion in the study of ischemic brain infarction in the rat, particularly as long as ischemic damage is delineated by TTC.