[Establishment and evaluation of a new method for determining hemodynamics of pulmonary hypertension rats].

Objective: By evaluating the hemodynamic parameters such as cardiac output (CO), right ventricular pressure (RVP), pulmonary artery pressure (PAP) and total pulmonary resistance index (TPRI) in pulmonary hypertension rat model, we established a more comprehensive hemodynamic evaluation system, which objectively evaluated the severity of disease and exercise tolerance in rats with pulmonary hypertension. Methods: SD rats were randomly divided into a control group and a model group with 5 rats in each group. The model group was intraperitoneally injected with SU5416 (20 mg/kg) and placed in an oxygen chamber at a 10% oxygen concentration for 21 days and then placed in a normoxic environment for 14 days. After modeling, rats were anesthetized and mechanically ventilated. The operator cut the skin along the right paraxial line, detached and ligated the intercostal artery, and then cut off the 3 and 4 ribs, exposing the heart and freeing aortic root about 0.2 cm. The flowmeter probe was set in the dissected aortic segment, and real-time recording time, blood flow waveforms, cardiac output were calculated accordingly. Then the needle attached to the baroreceptor was inserted into the right ventricle and the system acquired the right ventricular time-pressure waveform. After the waveform stabilized for about 30 seconds, the end of the cannula was sent to the pulmonary artery trunk through the entrance of the pulmonary artery to record the time-pressure curve of the pulmonary artery. Results: RVSP, PASP, PADP and mPAP in the model group were significantly higher than those of the control group [ RVSP(23.4±5.4) mmHg, 1 mmHg=0.133 kPa vs (56.4±13.0) mmHg, PASP (22.8±4.4) mmHg vs (58.5±14.9) mmHg, PADP (9.7±1.9) mmHg vs (30.3±7.0) mmHg, mPAP (14.1±2.7) mmHg vs (41.9±8.0) mmHg, all P<0.05 ]. Compared with the control group, the cardiac index in the model group was significantly lower [ CI (0.54±0.08) ml·min(-1)·g(-1) vs (0.40±0.09) ml·min(-1)·g(-1,) P=0.02 ]. Furthermore, compared with the control group, pulmonary vascular resistance index was significantly increased in the model group[PVRI (0.27±0.03) mmHg·ml(-1)·min(-1)·kg(-1) vs (0.06±0.01) mmHg·ml(-1)·min(-1)·kg(-1,) P<0.05]. The pathological results also showed that the middle part of pulmonary arterioles in the model group had muscular hypertrophy and muscular pulmonary arterioles, and even plexiform lesions. Conclusion: In this study, we established a new method that simultaneously determined several hemodynamic parameters such as RVSP, PASP, PADP, CO, CI and PVRI, which provided a more comprehensive assessment of hemodynamic changes in pulmonary hypertension rat models.

A nuclear-mitochondrial DNA interaction affecting hearing impairment in mice.

The pathophysiologic pathways and clinical expression of mitochondrial DNA (mtDNA) mutations are not well understood. This is mainly the result of the heteroplasmic nature of most pathogenic mtDNA mutations and of the absence of clinically relevant animal models with mtDNA mutations. mtDNA mutations predisposing to hearing impairment in humans are generally homoplasmic, yet some individuals with these mutations have severe hearing loss, whereas their maternal relatives with the identical mtDNA mutation have normal hearing. Epidemiologic, biochemical and genetic data indicate that nuclear genes are often the main determinants of these differences in phenotype. To identify a mouse model for maternally inherited hearing loss, we screened reciprocal backcrosses of three inbred mouse strains, A/J, NOD/LtJ and SKH2/J, with age-related hearing loss (AHL). In the (A/J x CAST/Ei) x A/J backcross, mtDNA derived from the A/J strain exerted a significant detrimental effect on hearing when compared with mtDNA from the CAST/Ei strain. This effect was not seen in the (NOD/LtJ x CAST/Ei) x NOD/LtJ and (SKH2/J x CAST/Ei) x SKH2/J backcrosses. Genotyping revealed that this effect was seen only in mice homozygous for the A/J allele at the Ahl locus on mouse chromosome 10. Sequencing of the mitochondrial genome in the three inbred strains revealed a single nucleotide insertion in the tRNA-Arg gene (mt-Tr) as the probable mediator of the mitochondrial effect. This is the first mouse model with a naturally occurring mtDNA mutation affecting a clinical phenotype, and it provides an experimental model to dissect the pathophysiologic processes connecting mtDNA mutations to hearing loss.

Mouse mutants from chemically mutagenized embryonic stem cells.

The drive to characterize functions of human genes on a global scale has stimulated interest in large-scale generation of mouse mutants. Conventional germ-cell mutagenesis with N-ethyl-N-nitrosourea (ENU) is compromised by an inability to monitor mutation efficiency, strain and interlocus variation in mutation induction, and extensive husbandry requirements. To overcome these obstacles and develop new methods for generating mouse mutants, we devised protocols to generate germline chimaeric mice from embryonic stem (ES) cells heavily mutagenized with ethylmethanesulphonate (EMS). Germline chimaeras were derived from cultures that underwent a mutation rate of up to 1 in 1,200 at the Hprt locus (encoding hypoxanthine guanine phosphoribosyl transferase). The spectrum of mutations induced by EMS and the frameshift mutagen ICR191 was consistent with that observed in other mammalian cells. Chimaeras derived from ES cells treated with EMS transmitted mutations affecting several processes, including limb development, hair growth, hearing and gametogenesis. This technology affords several advantages over traditional mutagenesis, including the ability to conduct shortened breeding schemes and to screen for mutant phenotypes directly in ES cells or their differentiated derivatives.

A new mouse mutant of the Cdh23 gene with early-onset hearing loss facilitates evaluation of otoprotection drugs.

We report a novel mutation (erlong, erl) of the cadherin 23 (Cdh23) gene in a mouse model for DFNB12 characterized by progressive hearing loss beginning from postnatal day 27 (P27). Genetic and sequencing analysis revealed a 208 T >C transition causing an amino-acid substitution (70S-P). Caspase expression was upregulated in mutant inner ears. Hearing was preserved (up to 35-dB improvement) in pan-caspase inhibitor Z-VAD-FMK-treated mutants compared with untreated mutants (P<0.05). Outer hair cell (OHC) loss in the cochleae of Z-VAD-FMK-treated mutants was significantly reduced compared with those of untreated mice. Thus, the erl mutation can lead to hearing loss through apoptosis. This is the first genetic mouse model of hearing loss shown to respond to otoprotective drug therapy. The short interval from initial hearing loss to deafness (P27-P90) makes this model ideal for screening and validating otoprotective drugs.

[Analysis on treatment of eight extremely severe burn patients in August 2nd Kunshan factory aluminum dust explosion accident].

Objective: To summarize the measures and experience of treatment in mass extremely severe burn patients. Methods: The clinical data and treatment of 8 extremely severe burn patients in August 2 Kunshan factory aluminum dust explosion accident who were admitted in the 100th Hospital of PLA on August 2nd, 2014, were retrospectively analyzed. There were 4 males and 4 females, aging 22-45 (34±7) years, with total burn area of 55%-98% [(89±15)%] total body surface area (TBSA) and full-thickness burn area of 45%-97% [(80±21)%] TBSA. All the 8 patients were accompanied with severe shock, inhalation injury, and blast injury. According to the requirements of former PLA General Logistics Department and Nanjing Military Command, a treatment team was set up including a special medical unit and a special care unit, with Chai Jiake from the First Affiliated Hospital of PLA General Hospital as the team leader, Zheng Qingyi from the 175th Hospital of PLA (the Affiliated Dongnan Hospital of Xiamen University) as the deputy leader, the 100th Hospital of PLA as the treatment base, and burn care, respiratory, nephrology, nursing specialists from the First Affiliated Hospital of PLA General Hospital, and the burn care experts and nursing staff from the 180th Hospital of PLA, 118th Hospital of PLA, 98th Hospital of PLA, and 175th Hospital of PLA, and nurses from the 85th Hospital of PLA, 455th Hospital of PLA, 101th Hospital of PLA, 113th Hospital of PLA as team members. Treatment strategies were adopted as unified coordination by the superior, unified responsibility of team leader, division of labor and cooperation between team members, and multidisciplinary cooperation led by department of burns. With exception of one patient who received deep vein catheterization before admission, the other 7 patients were treated with deep vein catheterization 0.5 to 3.0 hours after admission to correct hypovolemic shock as soon as possible. Eight patients received tracheotomy, and 7 patients were treated with mechanical ventilation by ventilator in protective ventilation strategy with low tide volume and low volume pressure to assist breathing. Fiberoptic bronchoscopy was done one to three times for all the 8 patients to confirm airway injuries and healing status. Escharectomy and Meek dermatoplasty in the extremities of all the 8 patients were performed 3 to 6 days after injury for the first time. Escharectomy, microskin grafting, and covering of large pieces of allogeneic skin on the trunks of 4 patients were performed 11 to 16 days after injury for the second time. The broad-spectrum antibiotics were uniformly used at first time of anti-infective therapy, and then the antibiotics species were adjusted in time. The balance of internal environment was maintained and the visceral functions were protected. One special care unit was on responsibility of only one patient. Psychological intervention was performed on admission. The rehabilitative treatment was started at early stage and in company with the whole treatment. Results: Acute renal injury occurred in 5 patients within 36 hours after injury and their renal function was restored to normal 4 days after injury due to active adjustment of fluid resuscitation program. No pulmonary complications, such as severe pulmonary infection and ventilator-associated pneumonia, occurred in the survived patients. One of the 8 patients died, and the other 7 patients were cured successfully. The wounds were basically healed in 2 patients in 26 or 27 days by 2 or 3 times of operation, and in 5 patients by 4 or 5 times of operation. The basic wound healing time was 26-64 (48±15) days for all the 7 patients. Conclusions: Treatment strategies of unified coordination by the superior, unified responsibility of team leader, division of labor and cooperation between team members, and multidisciplinary cooperation led by department of burns are the bases to successful treatment. Correcting shock as soon as possible is the prerequisite and closing wound as soon as possible is the key to successful treatment. Comprehensive treatment measures, such as maintaining and regulating the function of viscera, improving the body immunity, and preventing and treating the complications, are the important components to successful treatment. It is emphasized that in the treatment of mass extremely severe burn patients, specialist burn treatment should always be in the dominant position, and other related disciplines may play a part in auxiliary function.

A new spontaneous mutation in the mouse protocadherin 15 gene.

We have characterized a new allele of the protocadherin 15 gene (designatedPcdh15(av-6J)) that arose as a spontaneous, recessive mutation in the C57BL/6J inbred strain at Jackson Laboratory. Analysis revealed an inframe deletion in Pcdh15, which is predicted to result in partial deletion of cadherin domain (domain 9) in Pcdh15. Morphologic study revealed normal to moderately defective cochlear hair cell stereocilia in Pcdh15(av-6J) mutants at postnatal day 2 (P2). Stereocilia abnormalities were consistently present at P5 and P10. Degenerative changes including loss of inner and outer hair cells were seen at P20, with severe sensory cell loss in all cochlear turns occurring by P40. The hair cell phenotype observed in the 6J allele between P0 and P20 is the least severe phenotype yet observed in Pcdh15 alleles. However, young Pcdh15(av-6J) mice are unresponsive to auditory stimulation and show circling behavior indicative of vestibular dysfunction. Since these animals show severe functional deficits but have relatively mild stereocilia defects at a young age they may provide an appropriate model to test for a direct role of Pcdh15 in mechanotransduction.

Tauroursodeoxycholic acid prevents hearing loss and hair cell death in Cdh23(erl/erl) mice.

Sensorineural hearing loss has long been the subject of experimental and clinical research for many years. The recently identified novel mutation of the Cadherin23 (Cdh23) gene, Cdh23(erl/erl), was proven to be a mouse model of human autosomal recessive nonsyndromic deafness (DFNB12). Tauroursodeoxycholic acid (TUDCA), a taurine-conjugated bile acid, has been used in experimental research and clinical applications related to liver disease, diabetes, neurodegenerative diseases, and other diseases associated with apoptosis. Because hair cell apoptosis was implied to be the cellular mechanism leading to hearing loss in Cdh23(erl/erl) mice (erl mice), this study investigated TUDCA's otoprotective effects in erl mice: preventing hearing impairment and protecting against hair cell death. Our results showed that systemic treatment with TUDCA significantly alleviated hearing loss and suppressed hair cell death in erl mice. Additionally, TUDCA inhibited apoptotic genes and caspase-3 activation in erl mouse cochleae. The data suggest that TUDCA could be a potential therapeutic agent for human DFNB12.

Hearing loss associated with the modifier of deaf waddler (mdfw) locus corresponds with age-related hearing loss in 12 inbred strains of mice.

The modifier of deaf waddler (mdfw) and age-related hearing loss (Ahl) loci were both discovered as inbred strain polymorphisms that affect hearing loss in mice. Both loci map to the same position on chromosome (Chr) 10. The mdfw locus interacts epistatically with the deaf waddler (dfw) mutation on Chr 6, and the Ahl locus is a major contributor to AHL in several inbred strains. To investigate the possibility of allelism, we examined the correspondence of mdfw and Ahl phenotypes among 12 inbred mouse strains. The effects of strain-specific mdfw alleles on hearing loss were assessed in dfw2J/+ F1 hybrids produced from mating BALB-dfw2J/+ mice with mice from each of 12 inbred strains. F1 hybrids were then assessed for hearing by auditory-evoked brainstem response threshold analysis and classified as dfw2J/+ or +/+ by polymerase chain reaction typing. Heterozygosity for dfw2J accelerated hearing loss in F1 hybrids derived from all strains tested, except those produced with the B6.CAST + Ahl congenic strain. dfw2J/+ F1 hybrids derived from parental strains 129P1/ReJ, A/J, BUB/BnJ, C57BR/cdJ, DBA/2J, NOD/LtJ and SKH2/J exhibited a severe hearing loss by 12 weeks of age. Those derived from strains 129T2/SvEmsJ, C3H/HeJ, CBA/CaJ and NON/LtJ exhibited only a slight to intermediate hearing loss at that age. The hearing loss associated with these strain-specific mdfw alleles corresponds with previously determined Ahl allele effects, providing additional evidence that mdfw and Ahl are manifestations of the same gene. A functional relationship therefore may exist between the Ca2+ transporting activity of the dfw gene (Atp2b2) and AHL.

Assessment of hearing in 80 inbred strains of mice by ABR threshold analyses.

The common occurrence of hearing loss in both humans and mice, and the anatomical and functional similarities of their inner ears, attest to the potential of mice being used as models to study inherited hearing loss. A large-scale, auditory screening project is being undertaken at The Jackson Laboratory (TJL) to identify mice with inherited hearing disorders. To assess hearing sensitivity, at least five mice from each inbred strain had auditory brainstem response (ABR) thresholds determined. Thus far, we have screened 80 inbred strains of mice; 60 of them exhibited homogeneous ABR threshold values not significantly different from those of the control strain CBA/CaJ. This large database establishes a reliable reference for normal hearing mouse strains. The following 16 inbred strains exhibited significantly elevated ABR thresholds before the age of 3 months: 129/J, 129/ReJ, 129/SvJ, A/J, ALR/LtJ, ALS/LtJ, BUB/BnJ, C57BLKS/J, C57BR/cdJ, C57L/J, DBA/2J, I/LnJ, MA/MyJ, NOD/LtJ, NOR/LtJ, and SKH2/J. These hearing impaired strains may serve as models for some forms of human non-syndromic hearing loss and aid in the identification of the underlying genes.

A major gene affecting age-related hearing loss in C57BL/6J mice.

A major gene responsible for age-related hearing loss (AHL) in C57BL/6J mice was mapped by analyses of a (C57BL/6J x CAST/Ei) x C57BL/6J backcross. AHL, as measured by elevated auditory-evoked brainstem response (ABR) thresholds, segregated among backcross mice as expected for a recessive, primarily single-gene trait. Both qualitative and quantitative linkage analyses gave the same genetic map position for the AHL gene (Ahl on chromosome 10, near D10Mit5. Marker assisted selection was then used to produce congenic lines of C57BL/6J that contain different CAST-derived segments of chromosome 10. ABR test results and cochlear histopathology of aged progenitors of these congenic lines are presented. Ahl is the first gene causing late-onset, non-syndromic hearing loss that has been reported in the mouse.

Stent of shape-memory alloy for urethral obstruction caused by benign prostatic hyperplasia.

Dilation and positioning a stent in the prostatic urethra have become important alternatives for the management of benign prostatic hyperplasia (BPH), but both have significant drawbacks, namely the need to repeat the treatment in the former case and the conflict between the introducing means and the generation of sufficient expansile force in the latter case. A spiral of a Chinese titanium-nickel alloy with shape memory was implanted in 25 patients with BPH using a self-made coaxial sheath. With a follow-up of 3 to 20 months, the success rate is 92%. There has been no encrustation or migration of the spirals. We deem the spiral of this shape-memory alloy to be a good alternative in patients with BPH who are unfit for surgery.

Characterization of the triterpene saponins of the roots and rhizomes of blue cohosh (Caulophyllum thalictroides).

Seven triterpene saponins were isolated from n-butanol fractions of blue cohosh (Caulophyllum thalictroides) roots and rhizomes. Their structures were established by spectral ((1)H NMR, (13)C NMR, 2D-NMR, and APCI-MS) techniques and chemical reactions as hederagenin 3-O-alpha-L-arabinopyranoside (1); caulophyllogenin 3-O-alpha-L-arabinopyranoside (2); hederagenin 3-O-beta-D-glucopyranosyl-(1-->2)-alpha-L-arabinopyranoside (3); 3-O-alpha-L-arabinopyranosyl-hederagenin 28-O-alpha-L-rhamnopyranosyl-(1-->4)-beta-D-glucopyranosyl(1-->6)-beta-D-glucopyranoside (4); 3-O-alpha-L-arabinopyranosyl- caulophyllogenin 28-O-alpha-L-rhamnopyranosyl-(1-->4)-beta-D-glucopyranosyl(1-->6)-beta-D-glucopyranoside (5); 3-O-beta-D-glucopyranosyl-(1-->2)-alpha-L-arabinopyranosyl- echinocystic acid 28-O-alpha-L-rhamnopyranosyl-(1-->4)-beta-D-glucopyranosyl(1-->6)-beta-D-glucopyranoside (6); 3-O-beta-D-glucopyranosyl-(1-->2)-alpha-L-arabinopyranosyl-hederagenin 28-O-alpha-L-rhamnopyranosyl-(1-->4)-beta-D-glucopyranosyl(1-->6)-beta-D-glucopyranoside (7). All seven compounds were identified in this species for the first time.

Oxidative stress in aging in the C57B16/J mouse cochlea.

Presbycusis is a complex of high frequency hearing loss and disproportionate loss of speech discrimination that is seen concomitantly with physical signs of aging. Among the most extensively characterized strains of mice that show an early hearing loss is the C57B16/J strain, a strain that shows early onset of high frequency hearing loss at age 6 months and complete hearing loss by 1 year of age. The histopathology of this strain consists of loss of hair cells and spiral ganglion neurons in the basal turn, with a progression of loss of hair cells and ganglion neurons towards the apical portion of the cochlea as the animal ages. The process of aging has been extensively studied and although details differ in various organisms the consensus today is that oxidative stress, i.e. free radical-mediated tissue damage, is one of the core mechanisms of aging. Aerobic metabolism results in the creation of hydrogen peroxide and reactive oxygen species. These are normally detoxified by a variety of enzymes and free radical scavengers, including superoxide dismutase (SOD), catalase and glutathione. To determine whether oxidative stress plays a role in the pathophysiology of hearing loss in this mouse model of presbycusis we determined the relative change in mRNA production for selected free radical detoxifying enzymes in the C57B16/J mouse cochlea. Using semi-quantitative RT-PCR with tubulin mRNA as a control, relative levels of antioxidant enzyme mRNAs were determined. There was an overall increase in SOD1 mRNA levels when comparing 1 and 9 month time points, and a transient increase in the expression level of catalase mRNA. B6.CAST+ Ahl mice, which carry the C57B16/J genome but receive their Ahl gene from CAST mice, do not show these alteractions in antioxidant enzyme production. Our results suggest that at an age of 9 months, at which point significant hearing loss has developed, the C57B16/J mouse cochlea is exposed to increased levels of free radicals and that the Ahl gene of the C57B16/J mouse mediates this decrease in protective enzymes and therefore increase in levels of oxidative stress.

[Descending cord dorsum potentials evoked by stimulation at "Yong Quan" acupoint].

Segmental spinal cord potential (Y-sCDP) evoked by stimulation at "Yong Quan" acupoint of Sprague-Dawley rat vanished gradually when the recording electrode was moved rostrally along the dorsal surface of the spinal cord. When the recording electrode was placed more rostrally, for example at C6, another positive slow potential, Y-dCDP, could be recorded. Y-dCDP disappeared completely when the spinal cord had been sectioned at a higher cervical level. Electrolytic lesions of ventral periaqueductal gray (PAG) could also decrease remarkably the amplitude of Y-dCDP. This facet suggests that the PAG, an analgesia related nucleus, is involved in the generation of Y-dCDP induced by stimulation at "Yong Quan" acupoint.

[Inhibitory effect of esculentoside A on platelet activating factor released from calcimycin induced rat peritoneal macrophages].

Platelet activating factor (PAF) is a kind of inflammatory mediator. We used the method of aggregation of washed rabbit platelet to study the effect of esculentoside A (EsA) on the release of PAF from calcimycin (A23187)stimulated rat peritoneal macrophages and found that the release of PAF was inhibited by EsA in a time and dose dependent manner. The IC50 of EsA for the inhibition of PAF release was 1.5 mumol/L. Under the same condition, the release of PAF was also inhibited by mepacrine at 100 mumol/L. The results indicate that EsA is a potent inhibitor of PAF synthesis in rat peritoneal macrophages.

[Effects of Phytolacca acinosa polysaccharides II on lymphocyte proliferation and colony stimulating factor production from mice splenocytes in vitro].

Phytolacca acinosa polysaccharides II (PAP-II), a kind of polysaccharides isolated from Phytolacca acinosa Roxb with MW 40 kDa, on lymphocyte proliferation and colony stimulating factor (CSF) production from splenocytes in vitro, was studied. The radioactivities of [3H] TdR uptake by lymphocyte and bone marrow cells were used to determine the ability of lymphocyte proliferation and CSF production respectively. PAP-II was found to significantly augment splenocyte proliferation in a dose-dependent fashion, and significantly enhance Con A (1, 2.8 micrograms.ml-1) and LPS (3, 10, 30 micrograms.ml-1) induced lymphocyte proliferation at concentration of 31-125 micrograms.ml-1. As the concentration of PAP-II increased, significant suppression of Con A-induced lymphocyte proliferation was observed. Induction of CSF by PAP-II from splenocyte was confirmed at the present study. The optimal dosage was 100 micrograms.ml-1 and the optimal effect occurred on day 5. These results suggest that PAP-II can augment immunological function and enhance hematopoiesis.

[Leflunomide inhibits PAF induced DNA synthesis in rabbit synovial cells and PAF production from rat peritoneal macrophages].

Leflunomide (LFM, HWA 486) is an isoxazol derivative with antiphogistic and novel immunomodulating properties. It has been shown to be very effective in preventing and curing arthritis. In this report we found that platelet-activating factor (PAF) at 0.1-10 micrograms.ml-1 significantly stimulated DNA synthesis in cultured rabbit synovial cells. While LFM and its metabolite A771726 elicited inhibitory effects on this action of PAF. These two agents were also shown to markedly inhibit A23187 induced PAF production from rat peritoneal macrophages. The inhibition was dose and time-dependent. The inhibitory effects of LFM and A771726 on DNA synthesis in synovial cells and PAF production from macrophages may play an important role in the antiinflammatory effects of LFM.