Specific Pathogen-free Rats Research Articles

WenTong HuoXue Cream Can Inhibit the Reduction of the Pain-Related Molecule PLC-β3 in the Dorsal Root Ganglion of a Rat Model of Diabetic Peripheral Neuropathy.

WenTong HuoXue Cream (WTHX-Cream) has been shown to effectively alleviate clinical symptoms of diabetic peripheral neuropathy (DPN). This study investigated the gene and protein expression of the pain-related molecule PLC-β3 in the dorsal root ganglion (DRG) of DPN rats. 88 specific pathogen-free male Wistar rats were randomly divided into placebo (10 rats) and DPN model (78 rats) groups, and the 78 model rats were used to establish the DPN model by intraperitoneal injection of streptozotocin and were then fed a high-fat diet for 8 weeks. These rats were randomly divided into the model group, the high-, medium-, and low-dose WTHX-Cream + metformin groups, the metformin group, the capsaicin cream group, and the capsaicin cream + metformin group. After 4 weeks of continuous drug administration, the blood glucose, body weight, behavioral indexes, and sciatic nerve conduction velocity were measured. The pathological structure of the DRG and the sciatic nerve were observed. PLC-β3 mRNA and protein levels in the DRG of rats were measured. Compared with the model group, the high-dose WTHX-Cream group showed increased sciatic nerve conduction velocity, improved sciatic nerve morphological changes, and increased expression of PLC-β3 mRNA and protein in the DRG. This study showed that WTHX-Cream improves hyperalgesia symptoms of DPN by inhibiting the reduction of PLC-β3 mRNA and protein expression in the diabetic DRG of DPN rats.

Effects of dexmedetomidine on myocardium of rats with severe burn at early stage

Objective: To investigate the effects of dexmedetomidine on myocardium of rats at early stage after severe burn. Methods: Twenty specific pathogen free male SD rats were immersed in 90 ℃ hot water for 20 s, causing 30% total body surface area (TBSA) full-thickness scald (hereafter referred to as burn) on the back. And then they were divided into burn resuscitation group (BR) and burn resuscitation+ dexmedetomidine group (BRD) according to the random number table, with 10 rats in each group. Sodium lactate Ringer's solution (2 mL·kg(-1)·%TBSA(-1)) were intraperitoneally injected into rats of both groups after burn. Dexmedetomidine with dose of 1 μg/kg was intraperitoneally injected into rats of group BRD at the same time point. Another 5 rats in sham injury group (SI) were immersed in 37 ℃ water bath causing sham injury, and fluid resuscitation of rats in group SI was the same as that in group BR. Five rats of group BR and BRD were respectively selected at post burn hour (PBH) 6 and 24. And then left ventricular end-systolic internal diameter (LVIDs), left ventricular end-diastolic internal diameter (LVIDd), ejection fraction (EF), and cardiac output (CO) were determined with small animal ultrasonic imaging system. Plasma levels of cardiac troponin (cTn) I and cTnT were detected by enzyme-linked immunosorbent assay, and morphological changes of myocardium were observed under optical microscope and transmission electron microscope (observed only at PBH 24). In rats of group SI, morphological change of myocardium was observed at PBH 24, and the other indexes were detected as above. Data were processed with one-way analysis of variance and SNK test. Results: At PBH 6, EF value of rats in group BR [(98.0±2.8) %] was obviously higher than that in group SI [(91.0±0.4)%, P<0.05]. The other 3 cardiac ultrasound indexes of rats in group BR were close to those in group SI (with P values above 0.05). Each cardiac ultrasound index of rats between groups BRD and SI was close at PBH 6 (with P values above 0.05). At PBH 24, LVIDs levels of rats in group BR [(0.66±0.59) mm] and group BRD[(0.69±0.27) mm] were obviously lower than LVIDs level of rats in group SI [(1.65±0.33) mm, with P values below 0.05]. LVIDd, EF, and CO levels of rats were close among 3 groups at PBH 24 (with P values above 0.05). At PBH 6, the plasma levels of cTnI [(17.40±1.59) ng/mL] and cTnT [(1 488±229) pg/mL] of rats in group BR were significantly higher than those in group SI [(1.84±0.92) ng/mL and (169±12) pg/mL, with P values below 0.01]. At PBH 6 in group BRD, the plasma level of cTnI of rats [(2.58±0.60) ng/mL] was close to that in group SI (P>0.05), and the plasma level of cTnT [(649±190) pg/mL] was higher than that in group SI (P<0.01). At PBH 24, the plasma levels of cTnI and cTnT of rats in group SI were close to those in groups BR and BRD (with P values above 0.05). At PBH 24, the plasma level of cTnI of rats in group BRD was obviously lower than that in group BR (P<0.01). At PBH 6, the myocardial structures of rats in group BR and group BRD were normal, which were close to myocardial structure of rats in group SI at PBH 24. At PBH 24, obviously damaged myocardial tissue, disorderly arrangement of myofilament, and seriously damaged mitochondria were observed in rats of group BR, which were significantly ameliorated in rats of group BRD. Conclusions: Dexmedetomidine can protect the myocardium of rats with severe burn at early stage.

Angiotensin-converting enzyme 2 activation ameliorates pulmonary endothelial dysfunction in rats with pulmonary arterial hypertension through mediating phosphorylation of endothelial nitric oxide synthase

This study aims to investigate the effect of angiotensin-converting enzyme 2 (ACE2) activation on pulmonary endothelial function in the process of preventing pulmonary arterial hypertension (PAH) in rat models and to explore the underlying mechanisms. Specific pathogen free rats were randomly divided into five groups including control group, PAH group, PAH + Resorcinolnaphthalein (Res) group (ACE2 activation), PAH + Res + MLN4760 group (ACE2 inhibition), and PAH + Res + L-NAME group (endothelial nitric oxide synthase [eNOS] inhibition). Rat PAH model was constructed using combined left pneumonectomy with a single dose of monocrotaline injection 1 week after the surgery, and the rats were then given corresponding reagents. Hemodynamics, endothelial function, and pathologic changes were evaluated 3 weeks after monocrotaline injection. The concentration of nitric oxide (NO), expression of eNOS, and phosphorylation of eNOS at Ser1177 and Thr495 in the lung tissues from rats were also investigated.The Res-induced activation of ACE2 led to decreased mean pulmonary arterial pressure (mPAP) and pulmonary artery remodeling in the PAH + Res group comparing with the PAH rats (P < .05). In addition, the reduction in mPAP induced by acetylcholine (Ach) was augmented in PAH + Res group (P < .05), but this was not observed under the treatment with sodium nitroprusside (SNP) (P > .05). The ratio of decrease in mPAP caused by Ach to that caused by SNP (Ach/SNP) was also increased (P < .05) in ACE2-activated rats. However, the protective effects of ACE2 activation on PAH were counteracted by co-administration of MLN4760, an ACE2 antagonist (all P > .05). The mechanistic study showed that the concentration of NO in the lung tissues was downregulated in the PAH group but upregulated in the PAH + Res group (P < .05), whereas the NO concentration in the PAH + Res + MLN4760 group was not obviously different from that in the PAH group (P > .05). Regarding the factors regulating NO release, we found that the eNOS was upregulated in the PAH group, and Res did not affect the expression of eNOS. The phosphorylation of eNOS at Ser1177 was increased but at Thr495 was reduced after Res injection, when compared with the PAH group (P < .05). As expected, co-injection of MLN4760 eliminated these differences (P > .05). The reduction in mPAP induced by Ach was attenuated in the PAH + Res + L-NAME group compared with the PAH + Res group (P < .05), but this was not observed in rats treated with SNP (P > .05). The Ach/SNP ratio of decline in mPAP was also decreased in the PAH + Res + L-NAME group (P < .05). Activation of ACE2 had a protective role in the development of PAH via improving the function of pulmonary arterial endothelium. This effect was potentially mediated by promoted NO release as a consequence of increased phosphorylation of eNOS at Ser1177 and dephosphorylation of eNOS at Thr495.

Effects of different doses of docosahexaenoic acid in different doses on weight gain and lipid metabolism in neonatal rats during lactation

Objective To investigate the effect of docosahexaenoic acid (DHA) on the body weight growth and lipid metabolism of neonatal rats during lactation. Methods The specific pathogen free Sprague-Dawley neonatal rats were randomly assigned into 4 groups(high-dose group, medium-dose group, low-dose group and control group)by random number table method.The rats in 3 experiment groups received intragastric administration with DHA 600 mg/kg, 300 mg/kg and 100 mg/kg, respectively, while the control group were given 9 g/L saline, totally for 21 days.Body weight was monitored and compared among groups on postnatal day 1, 7, 14 and 21.And body weight growth rates at each time point were calculated.The serum concentrations of high density lipoprotein cholesterol(HDL-C), low density lipoprotein cholesterol(LDL-C), triglyceride(TG) and total cholesterol were measured and compared at 6-week and 8-week ages.The pathological and histological changes in the heart, the large vessel and the liver were observed at same time. Results The mean body weight of the neonatal rats were significantly different among 4 groups on postnatal day 7, 14 and 21 (F=17.334, 4.159, 6.485, all P 0.05). Compared with 2 groups, the mean body weight growth rate of the low-dose group was higher than that of the control group in the first week[(184.96±63.16)% vs.(141.02±72.07)%], but which was lower than that of the control group in the second week[(72.60±35.37)% vs.(103.20±40.11)%]; the medium-dose group was lower than the low-dose group at the first week[(116.78±51.59)% vs.(184.96±63.16)%], but higher than the low-dose group and lower than the control group at the second week[(139.93±67.4)% vs.(72.60±35.37)% and(103.20±40.11)%]; the high-dose group was lower than the low-dose group in the first week[(137.33±34.42)% vs.(184.96±63.16)%] and lower than that of the medium-dose group in the second week[(98.22±65.86)% vs.(139.93±67.4)%]; all these differences were statistically significant (all P 0.05) among 4 groups, but the mean concentration of HDL-C was statistically different (F=7.801, P 0.05), but the mean concentration of TG was significantly different (F=2.875, P<0.05). The mean concentration of TG in the medium-dose group was lower than that in the control group[(0.98±0.11) mmol/L vs.(1.36±0.09) mmol/L, P<0.05]. There were 5 (15.62%) neonatal rats in the high-dose group which were found to have adipose tissue accumulation around the large vessel walls and the heart and were confirmed by histological examination.The liver cells in these rats were found to have mild fatty changes.No similar changes were found in the other groups. Conclusions Neonatal rats supplemented with DHA during lactation can affect their body weight growth and lipid metabolism.Supplemented with high dose may bring risks, while moderate dose may bring benefits. Key words: Rat, newborn; Docosahexaenoic acid; Weight gain; Lipid

Effects and mechanisms of pirfenidone, prednisone and acetylcysteine on pulmonary fibrosis in rat idiopathic pulmonary fibrosis models

Context: Previous studies have reported that caveolin-1 (Cav-1) is associated with lung fibrosis. However, the role of Cav-1 expression in pirfenidone-treated idiopathic pulmonary fibrosis (IPF) is unknown. Objective: This study investigated Cav-1 expression in pirfenidone-treated IPF, and compared the effects of pirfenidone with acetylcysteine and prednisone on IPF. Materials and methods: Rat IPF model was established by endotracheal injection of 5 mg/kg bleomycin A5 into the specific pathogen-free Wistar male rats. Pirfenidone (P, 100 mg/kg once daily), prednisone (H, 5 mg/kg once daily) and acetylcysteine (N, 4 mg/kg 3 times per day) were used to treat the rat model by intragastric administration for 45 consecutive days, respectively. The normal rats without IPF were used as the controls. After 15, 30 and 45 days of drug treatment, lung histopathology was assessed. The expression of Cav-1 was determined using real-time quantitative PCR and Western blot; the expression of tumour necrosis factor-α (TNF-α), transforming growth factor-β1 (TGF-β1) and platelet-derived growth factor (PDGF) was determined by enzyme-linked immunosorbent assay. Results: After 15, 30 and 45 days of drug treatment, comparison of the three drug-treated groups with the model group showed significantly lower (p < 0.05) significance of airsacculitis and fibrosis scores of lung tissues, as well as expression of TGF-β1, TNF-α and PDGF, but the expression of Cav-1 was higher (p < 0.05). Compared with the N group, the fibrosis score was significantly lower and the protein expression of Cav-1 was significantly higher in the P group (p < 0.05). Additionally, the expression of Cav-1 was negatively correlated with the airsacculitis and fibrosis scores (r = −0.506, p < 0.01; r = -0.676, p < 0.01) as well as expression of TGF-β1, TNF-α and PDGF (r = −0.590, p < 0.01; r = −0.530, p < 0.01; r = −0.553, p < 0.01). Discussion and conclusion: Pirfenidone, prednisone and acetylcysteine can inhibit airsacculitis and pulmonary fibrosis in rat IPF models, which may be related with enhanced caveolin-1, reduced TNF-α, TGF-β1, PDGF.

Synchronous dynamic research on respiratory and intestinal microflora of chronic bronchitis rat model.

To investigate the mechanism of the Chinese medicine theory that Fei (Lung) and Dachang (Large Intestine) are exteriorly and interiorly related via synchronous observation on the dynamic changes of the respiratory and intestinal microflora. Forty specific pathogen free Sprague-Dawley rats were selected and randomly divided into blank (10 rats) and chronic bronchitis model groups (30 rats). The blank group rats were put into the smoke-free environment and the model group rats were put into the smoke environment in order to establish pulmonary disease (chronic bronchitis) model. Then the corresponding changes of the respiratory and intestinal microflflora of the model on 20th, 50th and 70th days were synchronously observed. The respiratory tract microflflora showed an increase in the total aerobic and Staphylococcus aureus and reduced anaerobic amount signifificantly on 20th day in the respiratory tract microflflora (P<0.05 or 0.01). On 50th day, total aerobic, total anaerobic amount and bififidobacterium signifificantly increased (P<0.05). On 70th day, Staphylococcus aureus reduced and lactobacillus increased signifificantly (P<0.01). The intestinal microflflora showed an increase in the total aerobic, Clostridium perfringens, enterobacter and enterococcus significantly increased on 20th day (P<0.05 or 0.01). Staphylococcus aureus on 50th day increased significantly (P<0.05). Total aerobic and enterococcus increased, total anaerobic and Clostridium perfringens reduced signifificantly on 70th day (P<0.05 or 0.01). The microecosystem of respiratory tract and intestine of rat model during the pathological process showed a dynamic disorder, indicating an interaction between the lung and large intestine which may be one of the connotations as they exteriorly and interiorly related.

Effects of reproductive technologies and SPF status on some physiological and behavioral characteristics in rats with arterial hypertension (ISIAH Strain)

Modern standards of Laboratory Animal Science include working with laboratory animals of a high quality, in particular, with specific pathogen-free (SPF) mice and rats. On the other hand, assisted reproductive technologies (ART) are widely used in modern medicine for human infertility treatment and genome resource banking. In the present study, a comparison of body weight, blood pressure (BP), and behavior in the elevated plus maze (EPM) test was made between three groups of ISIAH (inherited stress induced arterial hypertension) rats: a group of animals that were born and raised in a conventional animal facility and two groups from an SPF animal facility (animals born naturally and animals resulting from ART). There were no changes in BP between the groups, but the behavior of ISIAH rats differed depending on rearing conditions. In particular, the grooming time, as well as the number of defecations and the number of urinations during the test decreased in both groups of ISIAH rats born in the SPF animal facility as compared to ISIAH rats born in the conventional animal facility. The behavior of the ISIAH rat offspring resulting from ART was different from that of the naturally born group: the EPM test revealed reduced anxiety in the former. The results of the present study indicate that the rearing conditions and the reproductive technologies affect some behavioral characteristics in adult ISIAH rats, although they posessed arterial hypertension in all the conditions used in this study.

The rnc Gene Promotes Exopolysaccharide Synthesis and Represses the vicRKX Gene Expressions via MicroRNA-Size Small RNAs in Streptococcus mutans.

Dental caries is a biofilm-dependent disease that largely relies on the ability of Streptococcus mutans to synthesize exopolysaccharides. Although the rnc gene is suggested to be involved in virulence mechanisms in many other bacteria, the information regarding it in S. mutans is very limited. Here, using deletion or overexpression mutant assay, we demonstrated that rnc in S. mutans significantly positively regulated exopolysaccharide synthesis and further altered biofilm formation. Meanwhile, the cariogenecity of S. mutans was decreased by deletion of rnc in a specific pathogen-free (SPF) rat model. Interestingly, analyzing the expression at mRNA level, we found the downstream vic locus was repressed by rnc in S. mutans. Using deep sequencing and bioinformatics analysis, for the first time, three putative microRNA-size small RNAs (msRNAs) targeting vicRKX were predicted in S. mutans. The expression levels of these msRNAs were negatively correlated with vicRKX but positively correlated with rnc, indicating rnc probably repressed vicRKX expression through msRNAs at the post-transcriptional level. In all, the results present that rnc has a potential role in the regulation of exopolysaccharide synthesis and can affect vicRKX expressions via post-transcriptional repression in S. mutans. This study provides an alternative avenue for further research aimed at preventing caries.

Improved prescription of taohechengqi-tang alleviates D-galactosamine acute liver failure in rats.

To investigate the hepatoprotective effect of improved prescription of Taohechengqi-tang (IPTT) against acute liver failure (ALF) in rats. Seventy specific pathogen free male Wistar rats were randomly divided into four groups: control group (normal rats, n = 10), ALF group (ALF model, n = 20), Stronger Neo-Minophagen C (SNMC) group (ALF model + SNMC, n = 20), and IPTT group (ALF model + IPTT, n = 20). The ALF model group was administered an intraperitoneal injection of D-galactosamine (1.4 g/kg), and the control group received normal saline intraperitoneally. The SNMC and IPTT groups were treated with SMMC (15.6 mg/kg) or IPTT (28.6 g/kg) by gavage at 24 h intervals, and the ALF and control groups were treated with normal saline. At 36 h after injection, serum alanine aminotransferase, aspartate aminotransferase, total bilirubin, albumin, and cholinesterase and prothrombin time were determined, and liver histopathological scores were observed by microscopy after hematoxylin and eosin staining. mRNA expression of high mobility group box (HMGB) 1, toll-like receptor (TLR) 4, nuclear factor kappa B (NF-κB) and caspase-3 were analyzed via fluorescence quantitative reverse transcriptase polymerase chain reaction. Proliferating cell nuclear antigen (PCNA) immunohistochemistry in liver tissue was also performed. D-galactosamine notably decreased the biochemical and coagulation profiles in serum. IPTT not only improved liver function and histopathology but also normalized the gene expression levels in liver tissue. Compared with the model group, in the IPTT and SNMC groups, HMGB1 mRNA/β-actin (0.06 ± 0.03, 0.11 ± 0.04 vs 0.25 ± 0.04, P < 0.05); TLR4 mRNA/β-actin (0.07 ± 0.02, 0.22 ± 0.08 vs 0.41 ± 0.22, P < 0.05); NF-κB mRNA/β-actin (0.74 ± 0.41, 1.78 ± 0.64 vs 2.68 ± 1.35, P < 0.05); and caspase-3 mRNA/β-actin levels were all significantly reduced (1.61 ± 0.45, 2.57 ± 1.04 vs 3.41 ± 0.85, P < 0.05). The gene expression levels were significantly lower in the IPTT group than in the SNMC group (P < 0.05). Compared with the model group, the PCNA expression in liver tissue was significantly enhanced in the IPTT and SNMC groups (36.34 ± 4.91, 25.57 ± 2.94 vs 17.55 ± 2.40, P < 0.05). IPTT attenuates inflammation in ALF via inhibition of HMGB1 production, which may contribute to limited liver regeneration.

Therapeutic and immunoregulatory effect of GATA-binding protein-3/T-box expressed in T-cells ratio of astragalus polysaccharides on 2,4,6-trinitrobenzene sulfonic acid-induced colitis in rats.

To analyze the immunological characteristics of 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis model and examine the therapeutic effects and mechanisms of Astragalus polysaccharides (APS) treatment. Thirty-two male specific pathogen free Spragne-Dawley rats were randomly equally assigned to four groups: control, TNBS, APS and prednisone groups. Experimental colitis was induced by enema administration of TNBS. Then rats were treated with APS (0.5 g•kg-1•day-1, once daily) or prednisone (1.0 mg•kg-1•day-1, once daily) by gavage for 14 days. Macroscopic lesion and histological damage were determined, and activity of myeloperoxidase (MPO) was measured in the colonic tissues. Expressions of T-box expressed in T-cells (T-bet) and GATA-binding protein-3 (GATA-3) were determined by immunohistochemistry analysis and western blot. Both macroscopic lesion and histological colonic damage induced by TNBS were reduced by APS and prednisone treatment. These were accompanied by significant attenuation of MPO activity (P=0.03). TNBS intervention enhanced the expression of both GATA-3 and T-bet, but the expression of T-bet was significantly enhanced than that of GATA-3, resulting in significant reduction of GATA-3/T-bet ratio (P=0.025). APS administration enhanced the expression of T-bet (P=0.04) and GATA-3 (P=0.019) in comparison to TNBS group, and resulting in an up-regulated GATA-3/T-bet ratio. Prednisone treatment inhibited both expressions; however it also resulted in up-regulation of the GATA-3/T-bet ratio. These results demonstrated that APS exerted a beneficial immune regulatory effect on experimental colitis. It promoted the expression of T helper cell 1 (Th1) and T helper cell 2 (Th2) specific transcription factors but ultimately favor a shift toward Th2 phenotype, suggesting that APS possessed therapeutic potential in experimental colitis.

Preparation and analysis of active rat model of rheumatoid arthritis with features of TCM toxic heat-stasis painful obstruction

ObjectiveTo establish a collagen type II-induced rat model of rheumatoid arthritis (RA) presenting characteristics of the human form of the traditional Chinese syndrome pattern of toxic heat-stasis painful obstruction (bi zheng; arthromyodynia) as well as pathologic features of active RA. The Chinese herbal medicine Tengmei decoction was used to validate the animal model. MethodsNinety specific pathogen free Sprague-Dawley rats were randomly divided into a normal group of 6 rats and a model group of 84 rats. To establish the rat model of collagen-induced arthritis (CIA), bovine type II collagen in complete Freund's adjuvant was injected into the model group rats as a priming dose (Day 0) and boosting dose (Day 9). Changes in arthritic index (AI) scores, including limb swelling, were monitored. Thereafter, 24 successfully-established CIA rats were randomly assigned to 4 groups with 6 animals each: model, positive control drug, high-dose traditional Chinese herbal medicine, and traditional Chinese herbal medicine. A blank control group of 6 rats was included. After 12 weeks of intervention with Tengmei decoction, articular synovial tissue and serum specimens were collected to detect interleukin-2 (IL-2) and IL-17 transcription and protein expression levels. ResultsDays 0 through 9, the anterior–posterior and lateral diameters of the limbs in model group rats did not differ significantly when compared to the normal control group (P > .05). On day 16, anterior–posterior, lateral diameters, and footpad thicknesses of both hind limbs in the model group were higher than those of the normal control group (P < .01, P < .05). Total AI, AI of both hind limbs between Days 11 and 16 were significantly higher than those of the normal control group (P < .01). Real-time reverse transcription polymerase chain reaction analyses of rat joint tissues indicated that compared to the normal control group, levels of mRNA transcription of IL-2 and IL-17 were significantly upregulated (P < .01) in the model group. Compared with the model group, levels of mRNA transcription of the aforementioned inflammatory cytokines were significantly downregulated in the positive control drug and traditional Chinese medicine treatment groups (P < .01). Results of ELISA analyses indicated that compared with the normal control group, levels of protein expression of cytokines IL-2, IL-17 were significantly upregulated (P < .01) in the model group. Compared with the model group, levels of protein expression of the aforementioned inflammatory cytokines were significantly downregulated in the positive control drug and traditional Chinese medicine treatment groups (P < .01). ConclusionThe CIA model established in this study presents both active RA pathologic features and characteristics of the symptoms of toxic heat-stasis painful obstruction 12 weeks after successful establishment of an animal model. In addition, this study may be a valuable reference for development of animal studies with combined Eastern and Western medicines in dialectics and identification of diseases.

Reduced airway microbiota diversity is associated with elevated allergic respiratory inflammation

BackgroundAn increased prevalence of allergic disorders in developed countries has been associated with decreased exposure to environmental micro-organisms and an alteration of microbiota colonization. An appropriate model is needed to investigate the mechanisms by which hygiene environment-driven changes in microbiota could regulate allergic disorders. ObjectiveTo discover the correlation between the higher incidence and severity of allergies with the relative hygiene environment in a developed country. MethodsAllergic respiratory inflammation was induced in specific pathogen-free and control rats by sensitization and challenge with ovalbumin. The diversity of lower airway bacteria community was analyzed by polymerase chain reaction denaturing gradient gel electrophoresis and sequencing before ovalbumin sensitization. Allergic respiratory inflammation resulting in cellular infiltrate was measured after the last challenge. ResultsThe diversity of microbiota in the airway of specific pathogen-free rats decreased compared with the control rats; the more frequent microbiota in the control rats were Proteobacteria and Bacteroidetes. In addition, increased nasal rubbing and sneezing combined with exaggerated IgE production and leukocyte number was observed in ovalbumin-treated specific pathogen-free rats. ConclusionThese data indicate that the excessive “hygienic” environment resulted in a decreased bacterial diversity in the airway during infancy, leading to an increased susceptibility to allergic disease.

Comparison between animal temperature controller and artificial climate chamber employed for the establishment of classical heat

Objective To investigate the differences between animal temperature controller (ATC) and artificial climate chamber (ACC) used for the establishment of classical heat stroke (CHS) rat model. Methods Twenty-four male specific pathogen-free Wistar rats were randomly (random number) and equally assigned to three groups, namely room temperature control (C-C) group, heat stroke under conscious state (HS-C) group, and heat stroke under anesthesia (HS-A) group. Rats of HS-C or HS-A group were placed into ACC or ATC, then exposed to 35 ℃ heat stress. The systolic blood pressure (SBP) and core body temperature (Tc) were monitored. The time required for onset of HS was recorded. The white blood cell count (WBC) in peripheral blood and serum levels of C-reactive protein (CRP) , tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β) were measured. The histopathological changes of major organs were also confirmed by hematoxylin-eosin (HE) staining. Results The onset time in HS-A group was significantly shorter than that in HS-C group [ (40.0±4.3) min vs. (110.1±5.3) min, P 0.05) . However, there was no obvious difference in histopathological change in major organ observed between HS-A and HS-C groups. Conclusions In comparison of these two methods, ATC is similar to ACC in respect of the establishment of CHS rat model. ATC is quicker in onset of HS, and more simplified and economical than ACC and could substitute ACC. Key words: Heat stroke; Experiment animal model; Rat; Animal temperature controller; Artificial climate chamber

Effects of malathion on testicular spermatogenic function in rats

To investigate the effects of malathion on the testicular spermatogenic function of male rats and its working mechanism. Forty specific pathogen-free male Wistar rats were randomly and equally divided into four groups: three exposure groups and a control group. Malathion was administered orally to male rats in the exposure groups at 33.75, 54, and 108 mg/kg (1/32 LD₅₀, 1/20 LD₅₀, and 1/10 LD₅₀) for 60 days. Rats in the control group received an equal volume of water. The body weights of rats were measured after exposure. The organ weights and coefficients of the testes and epididymes were determined as soon as rats were sacrificed. The sperm motility, counts, and malformation rates were measured in the left epididymis. Histopathological changes, cell apoptosis, and the expression levels of Bcl-2/Bax in the testes of rats were observed using HE staining, terminal deoxynucleotidyl transferase-mediated dUPT-biotin nick end labeling, and immunohistochemistry SABC method. The body weights and the testis weights in the exposure groups were significantly lower than those in the control group (P < 0.01). The exposure groups had significantly lower sperm motility and significantly higher sperm malformation rates than the control group (P < 0.01). The sperm counts were significantly lower in the exposure groups than in the control group (P<0.01). The sperm counts and motility were negatively correlated with exposure dose (r = -0.81, P < 0.01; r = -0.51, P < 0.01), while the sperm malformation rate was positively correlated with exposure dose (r = 0.85, P 0.01). The exposure groups had significantly higher spermatogenic cell apoptosis rates than the control group (P<0.01). The expression level of Bax was significantly higher in the exposure groups than in the control group (P<0.01), while the expression level of Bcl-2 was significantly lower in the exposure groups than in the control group (P < 0.01). Histopathological examination of the testes showed degenerative changes in the seminiferous tubules at various doses along with the increase in malathion exposure dose. Malathion affects the testicular spermatogenic function of male rats and its working mechanism may involve cell apoptosis induced by down-regulation of Bcl-2 and up-regulation of Bax.

Impaired spatial learning and memory skill in Sprauge-Dawley rat model of congenital human cytomegalovirus infection

Objective To investigate the influence of congenital human cytomegalovirus (HCMV) infection on spatial learning and memory skill in rats and its mechanism. Methods Forty specific pathogen free Sprague-Dawley rats (12 weeks old, male and female mated by 1∶3) were selected and randomly divided into the experimental group and control group, fifteen in each group. Fertilized female rats in the experimental group were injected with 0.5 ml HCMV (1×10-6 of the 50% tissue culture infective dose/rat) by intraperitoneal inoculation on the third gestational day. At the same time, a 0.5 ml suspension of human embryonic lung fibroblast cells was inoculated intraperitoneally into fertilized female rats in the control group. HCMV-specific IgM and IgG in maternal peripheral blood of the two groups were detected on one week after inoculation. Both side of hippocampus of ten neonatal rats (five in each group), born by cesarean section, were collected for virus isolation. Spatial learning and memory skill in 4-6 week offspring in the two groups were assessed using the Morris Water Maze (MWM) test, and results were presented by Origin 8.0. Pathological damage in offspring was determined by microscopy and electron microscopy. Independent samples t-test, Chi-square and rank sum tests were used for statistical analysis. Results (1) Compared with the control group, litter size and mortality of neonatal rats within one week after birth in the experimental group were significantly higher [litter size: (8.7±3.1) vs (5.5±2.4), t=2.366, P=0.033; mortality of neonatal rats: 4.9% (6/122) vs 26.1% (18/69), χ2=191.020, P=0.000. In the MWM test, escape latency in 30-day old offspring in the experimental group was significantly increased compared with the control group during 4 days of training (F=499.473, P=0.000). The total distance in the experimental group was significantly increased compared with the control group (F=440.167, P=0.000). In addition, the numbers of platform crossing (4.21±1.44) and swimming time in the target quadrant [(26±4)%] in the congenital infection group were decreased compared with the control group [(7.50±1.72) and (47±5)%, t=7.182 and 15.487, P=0.003 and 0.000]. In the congenital infection group, liquefaction necrosis of the cerebral cortex (light microscopy) and myelin disintegration in the hippocampus (electron microscopy) were observed. Conclusions Injury of the cortex and hippocampus in offspring with congenital HCMV infection is involved in the pathological mechanism of decreased spatial learning and memory skill. Key words: Pregnancy complications, infectious; Cytomegalovirus infections; Hippocampus; Learning; Memory; Disease models, animal

Inhalation Toxicity of Ethylene Glycol in Rat

Several air fresheners and deodorants that contain ethylene glycol were found (1-2% (w/v)) in commercially available products. Ethylene glycol is a liquid used as a surfactant or an emulsifier in household products. The aim of this study is to determine the inhalation toxicity of ethylene glycol. The effect of whole-body inhalation to EG was performed for an inhalation study of rat. Specific pathogen-free male Sprague-Dawley rats (7 weeks old) were exposed to ethylene glycol in a stainless steel whole body inhalation chamber with a capacity of 1 m3 (Sibata Model VT3-X15 Japan). For acute and sub-acute rat inhalation toxicities, the differences of body weights were not statistically significant comparing with those of control rats. In sub-acute rat inhalation toxicity, the relative kidney weights were significantly higher in high-exposure group (500 mg/m3) than those of the other groups. In blood biochemistry the values of TG (triglyceride) and BUN (urea nitrogen) were significantly decreased. Other hematological changes with toxicological relevance were not observed in exposed male rats when compared with the control animals. The histopathological findings were not observed in the lung and kidney tissues exposed to chemicals comparing with those of control tissues. The No Observed Adverse Effect Level (NOAEL) of EG in 28 day’s inhalation test was evaluated to be over 100 mg/m3.

Haemotropic Mycoplasma Infection Revealed by Real-Time PCR in Specific Pathogen-Free Rats

Abstract The presence of Mycoplasma haemomuris (haemoplasma) in blood samples collected from specific pathogen-free (SPF) laboratory rats bred in Japan was reported. Its presence was examined in Fischer 344, Sprague-Dawley (SD), and Wistar rat strains of both sexes by real-time PCR. All strains were positive for M. haemomuris infection. The 16S rRNA gene of M. haemomuris strain detected in the animals was amplified using end-point PCR. Only the entire nucleotide sequence of 16S rRNA gene of a mycoplasma strain detected in SD rats was determined and compared to those of other haemoplasmas. Our investigations suggest a wide M. haemomuris infection among the SPF rats purchased from commercial breeders in Japan.

Effect of melatonin on p38MAPKsignaling pathway in rats with phosgene-induced lung injury

To investigate the effect of melatonin (MT) on p38 mitogen-activated protein kinase (MAPK) signaling pathway in rats with phosgene-induced lung injury. Fifty specific pathogen-free male Sprague-Dawley rats were randomly divided into phosgene inhalation group, air control group, saline control group, MT treatment group, and SB203580 (specific inhibitor of p38 MAPK) group, with 10 mice in each group. All groups except the air control group were exposed to phosgene, and the animals were sacrificed 6 h later. Lung wet/dry weight (W/D) ratio and the content of malondialdehyde (MDA) and nitric oxide (NO) and activity of myeloperoxidase (MPO) in bronchoalveolar lavage fluid (BALF) were measured. The qualitative and quantitative expression of p38 MAPK and phospho-p38 MAPK (p-p38) was measured by immunohistochemistry (IHC) and Western blot, respectively. Inducible nitric oxide synthase (iNOS) level in lung tissue was determined by Western blot. Compared with the air control group, the phosgene inhalation group had significantly increased lung W/D ratio and neutrophil count in BALF (P < 0.01); the MT treatment group had significantly lower neutrophil count and lung W/D ratio than the phosgene inhalation group (P < 0.05). IHC demonstrated that the air control group had relatively weak expression of p-p38 in lung tissue; the expression of p-p38 was significantly up-regulated after phosgene inhalation, and it was mainly distributed in infiltrating inflammatory cells and vascular endothelial cells, positive in the cytoplasm and nucleus of many cells. The distribution of p-p38-positive cells in the MT treatment and SB203580 groups was similar to that in the phosgene inhalation group, but the MT treatment and SB203580 groups had a significantly reduced number of cells with p-p38-positive nuclei and a significantly reduced intensity of p-p38 expression signals. The phosgene inhalation group had significantly increased content of MDA and NO and activity of MPO compared with the air control group (P < 0.01); the MT treatment and SB203580 groups had significantly reduced content of MDA and NO and activity of MPO compared with the phosgene inhalation group (P < 0.05), but had higher content of MDA and NO and activity of MPO than the air control group. The Western blot showed that the phosgene inhalation group had significantly increased expression of iNOS and p-p38 compared with the air control group (P < 0.01); the MT treatment and SB203580 groups had lower expression of iNOS and p-p38 than the phosgene inhalation group (P < 0.05). MT and SB203580 have a significant protective effect in rats with phosgene-induced lung injury, and the mechanism may be associated with scavenging free radicals and inhibiting activation of p38 MAPK and expression of iNOS.

Absence of the gut microbiota enhances anxiety-like behavior and neuroendocrine response to acute stress in rats

Establishment of the gut microbiota is one of the most important events in early life and emerging evidence indicates that the gut microbiota influences several aspects of brain functioning, including reactivity to stress. To better understand how the gut microbiota contributes to a vulnerability to the stress-related psychiatric disorders, we investigated the relationship between the gut microbiota, anxiety-like behavior and HPA axis activity in stress-sensitive rodents. We also analyzed the monoamine neurotransmitters in the brain upper structures involved in the regulation of stress and anxiety. Germfree (GF) and specific pathogen free (SPF) F344 male rats were first subjected to neurological tests to rule out sensorimotor impairments as confounding factors. Then, we examined the behavior responses of rats to social interaction and open-field tests. Serum corticosterone concentrations, CRF mRNA expression levels in the hypothalamus, glucocorticoid receptor (GR) mRNA expression levels in the hippocampus, and monoamine concentrations in the frontal cortex, hippocampus and striatum were compared in rats that were either exposed to the open-field stress or not. GF rats spent less time sniffing an unknown partner than SPF rats in the social interaction test, and displayed a lower number of visits to the aversive central area, and an increase in latency time, time spent in the corners and number of defecations in the open-field test. In response to the open-field stress, serum corticosterone concentrations were 2.8-fold higher in GF than in SPF rats. Compared to that of SPF rats, GF rats showed elevated CRF mRNA expression in the hypothalamus and reduced GR mRNA expression in the hippocampus. GF rats also had a lower dopaminergic turnover rate in the frontal cortex, hippocampus and striatum than SPF rats. In stress-sensitive F344 rats, absence of the gut microbiota exacerbates the neuroendocrine and behavioral responses to acute stress and the results coexist with alterations of the dopaminergic turnover rate in brain upper structures that are known to regulate reactivity to stress and anxiety-like behavior.

Protective effects of traditional Chinese medicine formula NaoShuanTong capsule on haemorheology and cerebral energy metabolism disorders in rats with blood stasis

NaoShuanTong capsule (NSTC), an oral traditional Chinese medicine formula, is composed of Pollen Typhae, Radix Paeoniae Rubra, Rhizoma Gastrodiae, Radix Rhapontici and Radix Curcumae. It has been widely used to treat ischemic stroke in clinic for many years in China. In addition to neuronal apoptosis, haemorheology and cerebral energy metabolism disorders also play an important role in the pathogenesis and development of ischemic stroke. The present study was designed to evaluate the in vivo protective effects of NSTC on haemorheology and cerebral energy metabolism disorders in rats with blood stasis. Sixty specific pathogen-free sprague-dawley rats, male only, were randomly divided into six groups (control group, model group, aspirin (100 mg/kg/d) group, NSTC low-dose (400 mg/kg/d) group, NSTC intermediate-dose (800 mg/kg/d) group, NSTC high-dose (1600 mg/kg/d) group) with 10 animals in each. The rats except those in the control group were placed in ice-cold water (0–4 °C) for 5 min during the time interval (4 h) of two adrenaline hydrochloride injections (0.8 mg/kg) to induce blood stasis. After treatment, whole blood viscosity at three shear rates, plasma viscosity and erythrocyte sedimentation rate significantly decreased in NSTC intermediate- and high-dose groups; erythrocyte aggregation index and red corpuscle electrophoresis index significantly decreased in all the three dose NSTC groups. Moreover, treatment with high-dose NSTC could significantly improve Na+–K+ adenosine triphosphatase (ATPase) and Ca2+ ATPase activity, as well as lower lactic acid level in brain tissues. These results demonstrated the protective effects of NSTC on haemorheology and cerebral energy metabolism disorders, which may provide scientific information for the further understanding of mechanism(s) of NSTC as a clinical treatment for ischemic stroke. Furthermore, the protective effects of activating blood circulation as observed in this study might create valuable insight for the utilisation of NSTC to be a feasible alternative therapeutic agent for patients with blood stasis.