Four-parameter analysis in modified Rotarod test for detecting minor motor deficits in mice.

BACKGROUND: The Rotarod test with commercial apparatus is widely used to assess locomotor performance, balance and motor learning as well as the deficits resulting from diverse neurological disorders in laboratory rodents due to its simplicity and objectivity. Traditionally, the test ends when rodents drop from the accelerating, turning rod, and the only parameter used commonly is "latency to fall". The values of individual animals can often vary greatly. RESULTS: In the present study, we established a procedure for mice with 4 consecutive days of training with 4 trials per day and modified the testing procedure by placing the mice back on the rod repeatedly after each fall until the trial ends (5 min). Data from the fourth training day as baseline results showed that the second, third and fourth trial were more consistent than the first, probably due to habituation or learning. There was no difference between the second, third and fourth trial, two trials may be sufficient in testing. We also introduced 3 additional read-outs: Longest duration on the rod (s), Maximal distance covered (cm), and Number of falls to better evaluate the motor capacity over the 5 min of testing. We then used this 4-parameter analysis to capture the motor deficits of mice with mild to moderate traumatic brain injuries (by a weight dropping on the skull (Marmarou model)). We found that normalization of data to individual baseline performance was needed to reduce individual differences, and 4 trials were more sensitive than two to show motor deficits. The parameter of Maximal distance was the best in detecting statistically significant long-term motor deficits. CONCLUSIONS: These results show that by making adjustments to the protocol and employing a more refined analysis, it is possible to expand a widely used routine behavioral test with additional accessible parameters that detect relevant deficits in a model of mild to moderate traumatic brain injury. The modified Rotarod test maybe a valuable tool for better preclinical evaluations of drugs and therapies.

Development of a Sensitive Enzyme-Linked Immunosorbent Assay with Three Amplification Antibodies to Detect Low-Abundant Mouse Antibodies in the Mouse Central Nervous System.

Recombinant Abs are gaining increasing importance for the treatment of certain cancers or immunological or neurologic disorders. The ELISA is one of the most used analytical tools for detecting and quantifying Abs of interest. However, the performance of ELISAs often varies because of nonstandard experimental procedures as well as inadequate data analysis. In our study, we standardized a procedure and statistical analysis for a highly sensitive ELISA of a mouse Ab in mouse (C57BL/6J) CNS tissue. The following steps are of crucial importance: 1) calculation of the limit of detection based on control tissue lysate samples in the same testing buffer as the testing samples; 2) calculation of the limit of quantification as measured with acceptable accuracy and precision; and 3) a five-parameter logistic regression model to interpolate the symmetric and asymmetric standard curves. We also show that three amplification Abs can significantly increase the sensitivity of the ELISA compared with a two amplification Ab setup. This standardized procedure may be a valuable tool to increase the sensitivity, reproducibility, and precision of ELISA studies in basic science and translational research.

Farnesoid X receptor knockout protects brain against ischemic injury through reducing neuronal apoptosis in mice.

BACKGROUND: Farnesoid X receptor (FXR) is a nuclear receptor that plays a critical role in controlling cell apoptosis in diverse diseases. Previous studies have shown that knocking out FXR improved cardiac function by reducing cardiomyocyte apoptosis in myocardial ischemic mice. However, the role of FXR after cerebral ischemia remains unknown. In this study, we explored the effects and mechanisms of FXR knockout (KO) on the functional recovery of mice post cerebral ischemia-reperfusion. METHODS: Adult male C57BL/6 wild type and FXR KO mice were subjected to 90-min transient middle cerebral artery occlusion (tMCAO). The mice were divided into five groups: sham, wild-type tMCAO, FXR KO tMCAO, wild-type tMCAO treated with calcium agonist Bayk8644, and FXR KO tMCAO treated with Bayk8644. FXR expression was examined using immunohistochemistry and Western blot. Brain infarct and brain atrophy volume were examined at 3 and 14 days after stroke respectively. Neurobehavioral tests were conducted up to 14 days after stroke. The protein levels of apoptotic factors (Bcl-2, Bax, and Cleaved caspase-3) and mRNA levels of pro-inflammatory factors (TNF-α, IL-6, IL-1ß, IL-17, and IL-18) were examined using Western blot and RT-PCR. TUNEL staining and calcium imaging were obtained using confocal and two-photon microscopy. RESULTS: The expression of FXR was upregulated after ischemic stroke, which is located in the nucleus of the neurons. FXR KO was found to reduce infarct volume and promote neurobehavioral recovery following tMCAO compared to the vehicle. The expression of apoptotic and pro-inflammatory factors decreased in FXR KO mice compared to the control. The number of NeuN+/TUNEL+ cells declined in the peri-infarct area of FXR KO mice compared to the vehicle. We further demonstrated that inhibition of FXR reduced calcium overload and addition of ionomycin could reverse this neuroprotective effect in vitro. What is more, in vivo results showed that enhancement of intracellular calcium concentrations could aggravate ischemic injury and reverse the neuroprotective effect of FXR KO in mice. CONCLUSIONS: FXR KO can promote neurobehavioral recovery and attenuate ischemic brain injury, inflammatory release, and neuronal apoptosis via reducing calcium influx, suggesting its role as a therapeutic target for stroke treatments.

L-glutamine protects mouse brain from ischemic injury via up-regulating heat shock protein 70.

INTRODUCTION: L-glutamine is an antioxidant that plays a role in a variety of biochemical processes. Given that oxidative stress is a key component of stroke pathology, the potential of L-glutamine in the treatment of ischemic stroke is worth exploring. AIMS: In this study, we investigated the effect and mechanisms of action of L-glutamine after cerebral ischemic injury. RESULTS: L-glutamine reduced brain infarct volume and promoted neurobehavioral recovery in mice. L-glutamine administration increased the expression of heat-shock protein 70 (HSP70) in astrocytes and endothelial cells. Such effects were abolished by the coadministration of Apoptozole, an inhibitor of the ATPase activity of HSP70. L-glutamine also reduced oxidative stress and neuronal apoptosis, and increased the level of superoxide dismutase, glutathione, and brain-derived neurotrophic factor. Cotreatment with Apoptozole abolished these effects. Cell culture study further revealed that the conditioned medium from astrocytes cultured with L-glutamine reduced the apoptosis of neurons after oxygen-glucose deprivation. CONCLUSION: L-glutamine attenuated ischemic brain injury and promoted functional recovery via HSP70, suggesting its potential in ischemic stroke therapy.

Identification of homocysteine-suppressive mitochondrial ETC complex genes and tissue expression profile - Novel hypothesis establishment.

Hyperhomocysteinemia (HHcy) is an independent risk factor for cardiovascular disease (CVD) which has been implicated in matochondrial (Mt) function impairment. In this study, we characterized Hcy metabolism in mouse tissues by using LC-ESI-MS/MS analysis, established tissue expression profiles for 84 nuclear-encoded Mt electron transport chain complex (nMt-ETC-Com) genes in 20 human and 19 mouse tissues by database mining, and modeled the effect of HHcy on Mt-ETC function. Hcy levels were high in mouse kidney/lung/spleen/liver (24-14 nmol/g tissue) but low in brain/heart (~5 nmol/g). S-adenosylhomocysteine (SAH) levels were high in the liver/kidney (59-33 nmol/g), moderate in lung/heart/brain (7-4 nmol/g) and low in spleen (1 nmol/g). S-adenosylmethionine (SAM) was comparable in all tissues (42-18 nmol/g). SAM/SAH ratio was as high as 25.6 in the spleen but much lower in the heart/lung/brain/kidney/liver (7-0.6). The nMt-ETC-Com genes were highly expressed in muscle/pituitary gland/heart/BM in humans and in lymph node/heart/pancreas/brain in mice. We identified 15 Hcy-suppressive nMt-ETC-Com genes whose mRNA levels were negatively correlated with tissue Hcy levels, including 11 complex-I, one complex-IV and two complex-V genes. Among the 11 Hcy-suppressive complex-I genes, 4 are complex-I core subunits. Based on the pattern of tissue expression of these genes, we classified tissues into three tiers (high/mid/low-Hcy responsive), and defined heart/eye/pancreas/brain/kidney/liver/testis/embryonic tissues as tier 1 (high-Hcy responsive) tissues in both human and mice. Furthermore, through extensive literature mining, we found that most of the Hcy-suppressive nMt-ETC-Com genes were suppressed in HHcy conditions and related with Mt complex assembly/activity impairment in human disease and experimental models. We hypothesize that HHcy inhibits Mt complex I gene expression leading to Mt dysfunction.

[Experimental study of glucocorticoid in the treatment of acute respiratory distress syndrome induced by E.coli].

OBJECTIVE: To evaluate the effect of glucocorticoid (GC) in the treatment of septic acute respiratory distress syndrome (ARDS). METHODS: ARDS model was reproduced by intraperitoneal injection of E.coli in piglets. Nine male piglets were randomly divided into control (C group), early (GC1) and middle (GC2) stage treatment groups. In the latter two groups methylprednisolone 20 mg was intravenously,given every 12 hours (4 mg(-1)xkg(-1)xd(-1)). All of the clinical data and survival time during 72 hours were collected and analyzed, including the collection of bronchioalveolar lavage fluid (BALF) for the determination of total protein (TP), total phospholipids (TPL), disaturated phosphatidyl choline (DSPC), and measurement of alveolar tension. Ratio of pulmonary wet and dry(W/D) weight was determined routinely, and pathological changes and their severity were evaluated by optical microscope and Smith scores. RESULTS: ARDS model was reproduced at (8.3+/-8.5) hours, and survival time of three groups was (11.0+/-6.6) hours, (35.3+/-12.5) hours and (52.5+/-13.8) hours respectively. Animals in GC1 and GC2 survived longer than those in C group, but there was no statistically significant difference among them (both P>0.05).Oxygen index (PaO(2)/FiO(2)) and mean arterial pressure (MAP) were improved in GC1 much better than those of controls (both P<0.05), and the same was true in GC2 as compared that before GC treatment (P<0.05 or P<0.01). There were no significant differences among TPL, DSPC, white blood cell count (WBC) of BALF in each group, so were lung surface tension and W/D (all P>0.05). TP of BALF was significantly higher in GC1 than that in GC2. Compared with C group, alveolar and interstitial edema, inflammation and hemorrhage were more severe in GC1 (all P<0.01), hyaline membrane was less(P<0.01) and no difference in atelectasis (P>0.05). The alveolar and interstitial edema and inflammation in GC2 were more severe than in C group(all P<0.01), and there was no difference in hemorrhage and hyaline membrane formation (both P>0.05). CONCLUSION: GC might improve hypoxemia and septic shock as a result of septic ARDS. But the treatment has no influence on pulmonary surfactant (PS), surface tension and lung pathology of ARDS in piglets challenged with intraperitoneal E.coli.

[Evaluation of glucocorticoid in treatment for patients with acute respiratory distress syndrome as a result of serious community-acquired pneumonia].

OBJECTIVE: To evaluate the usage of glucocorticoid (GC) in treatment for patients with acute respiratory distress syndrome (ARDS) resulting from serious community-acquired pneumonia (SCAP). METHODS: The clinical data from all patients with ARDS resulting from SCAP in medical ICU (MICU) from May 2000 to Feb. 2003 were collected. Their age, sex, acute physiology and chronic health evaluation (APACHE II) score, PaO(2)/FiO(2) and Qs/Qt, the severity of SCAP, mechanical ventilation (MV) and the level of positive end-expiratory pressure (PEEP), time of stay in ICU, improvement of SCAP and oxygenation, as well as mortality and reasons of death were analyzed, respectively. So was did the influence of administration of GC on hypoxemia, septic shock, and their prognosis. RESULTS: There were 24 cases totally, among them 7 patients had not taken GC, and 5 patients were cured (71.4 percent), and the other 2 cases died (28.6 percent) Their direct cause of death were multiple organ dysfunction syndrome (MODS) and ARDS, respectively In 17 cases GC was given because hypoxemia and septic shock could not be alleviated with ordinary therapy, including MV Among them only 5 patients (29.4 percent) were cured, and all others (12 cases, 70.6 percent) died, and the major direct cause of death was MODS (6 cases, 75.0 percent). A few of them died of ARDS and septic shock (1 case, 12.5 percent, respectively). The severity of SCAP, as well as other clinical data of the survivors, showed no significant difference compared with the nonsurvivors (P>0.05). But except for their PaO2/FiO2, Qs/Qt and shock, their pulmonary infection was better controlled than deaths (P<0.001). CONCLUSION: Refractory hypoxemia and septic shock of patients with pulmonary ARDS might be alleviated by GC when they are treated with routine methods, including MV, thus it enabled to win the time for other effective treatments.

[Evaluation of glucocorticoid in treatment for patients with acute respiratory distress syndrome].

OBJECTIVE: To value of glucocorticoid (GC) in treatment for patients with acute respiratory distress syndrome (ARDS) was evaluated. METHODS: The clinical data from all patients with ARDS in medical ICU (MICU) during May 2000 to Aug. 2002 were collected. They were divided into two groups, GC and non-GC groups, in order to compare their age, sex, acute physiology and chronic health evaluation II (APACHE II) score, PaO2/FiO2, Qs/Qt, level of positive end-expiratory pressure(PEEP), mortality and dead reason of death, depending on whether GC was given or not. In cases with administration of GC, the dosage, as well as duration of treatment was analyzed in terms of the overcome. RESULTS: There were 77 cases totally, among them 60 cases were of GC group and 17 of non -GC. Their age, sex, APACHE II score, PaO2/FiO2, Qs/Qt, use of artificial ventilation and its duration, level of PEEP, and the extent of relief from hypoxemia showed no significant differences between two groups (P>0.05). Even the mortality for patients who were treated with GC was higher than those without (71.7% vs. 52.9%), though there was no statistically significant difference (P>0.05). The percentage of patients died primarily of ARDS was low in both groups (7.0% and 11.1%). The age, APACHE II score and underlying diseases for non-survivors were older and higher than survivors (P<0.001 or P<0.005) and their duration of staying in ICU was shorter than the latter (P<0.05). The mortality of patients who were given GC before or during 24 hours of the establishment of the diagnosis of ARDS (66.7% and 68.2%) was lower than those who were given GC 24 hours after the diagnosis of ARDS (90.0%). CONCLUSION: GC could be one of effective treatments for ARDS, and it should be given without hesitation when refractory hypoxemia and shock were found.