Prevalence of Bacterial Coinfections with Vibrio harveyi in the Industrialized Flow-through Aquaculture Systems in Hainan Province: A Neglected High-Risk Lethal Causative Agent to Hybrid Grouper.

Vibrio harveyi is one of the most serious bacterial pathogens to aquatic animals worldwide. Evidence is mounting that coinfections caused by multiple pathogens are common in nature and can alter the severity of diseases in marine animals. However, bacterial coinfections involving V. harveyi have received little attention in mariculture. In this study, the results of pathogen isolation indicated that bacterial coinfection was a common and overlooked risk for hybrid groupers (♀ Epinephelus polyphekadion × â™‚ E. fuscoguttatus) reared in an industrialized flow-through pattern in Hainan Province. The artificial infection in hybrid groupers revealed that coinfections with V. harveyi strain GDH11385 (a serious lethal causative agent to groupers) and other isolated pathogens resulted in higher mortality (46.67%) than infection with strain GDH11385 alone (33.33%), whereas no mortality was observed in single infection with other pathogens. Furthermore, the intestine, liver and spleen of hybrid groupers are target organs for bacterial coinfections involving V. harveyi. Based on the infection patterns found in this study, we propose that V. harveyi may have a specific spatiotemporal expression pattern of virulence genes when infecting the host. Taken together, bacterial coinfection with V. harveyi is a neglected high-risk lethal causative agent to hybrid groupers in the industrialized flow-through aquaculture systems in Hainan Province.

[Distribution Characteristics, Ecological Risk Assessment, and Source Tracing of Heavy Metals in the Sediments of Typical Lakes in the Middle Reaches of the Yangtze River].

In this study, surface sediment samples were collected from Dongting Lake, Honghu Lake, and Chihu Lake, and the concentrations of 10 heavy metals were measured. Then, the potential risk of heavy metal accumulation was evaluated using the cumulative pollution index (Igeo), the enrichment factor (EF), and the potential ecological risk index (RI), and the sources were traced using correlation analysis (Pearson) and principal component analysis (PCA). The results showed that the pollution and potential ecological risk of Cd were the most serious. The mean values of Cd in East Dongting Lake, Honghu Lake, and Chihu Lake were 2.85, 1.59, and 3.57 mg·kg-1, respectively. The concentrations of Cd were 25.87, 11.36, and 37.58 times higher than the soil background values of the corresponding provinces, which exceeded the risk screening value (0.6 mg·kg-1). Particularly, the Cd concentration of Chihu Lake exceeded the risk control value (3.0 mg·kg-1). Besides Cd, the concentration of As in Honghu Lake was also of concern. At the same time, the Cu, As, Zn, and Pb in Chihu Lake should not be neglected. The potential ecological risks of the three lakes were ranked as follows:Chihu Lake (RI=1 127)>East Dongting Lake (RI=831)>Honghu Lake (RI=421). The primary sources of heavy metals were industrial mining, agricultural production, and aquaculture, and some heavy metals (Mn and Cu) were from natural sources. This study was of great significance for the prevention and control of heavy metals in the sediments of typical lakes in the middle reaches of the Yangtze River.

Development of biodegradable polycaprolactone film as an internal fixation material to enhance tendon repair: an in vitro study.

BACKGROUND: Current tendon repair techniques do not provide sufficient tensile strength at the repair site, and thus early active motion rehabilitation after tendon repair is discouraged. To enhance the post-operative tensile strength, we proposed and tested an internal fixation technique using a polycaprolactone (PCL) biofilm. PCL was chosen for its good biocompatibility, excellent mechanical strength, and an appropriate degradation time scale. METHODS: PCL biofilms were prepared by a modified melt-molding/leaching technique, and the physical and mechanical properties and in vitro degradation rate were assessed. The pore size distribution of the biofilm and the paratenon of native tendons were observed using scanning electron microscopy. Next, we determined whether this biofilm could enhance the tensile strength of repaired tendons. We performed tensile tests on rabbit Achilles tendons that were first lacerated and then repaired: 1) using modified Kessler suture combined with running peripheral suture ('control' group), or 2) using biofilm to wrap the tendon and then fixation with sutures ('biofilm' group). The influence of different repair techniques on tendon tensile strength was evaluated by mechanical testing. RESULTS: The novel biofilm had supple texture and a smooth surface. The mean thickness of the biofilm was 0.25 mm. The mean porosity of the biofilm was 45.3%. The paratenon of the rabbit Achilles tendon had pores with diameters ranging from 1 to 9 µm, which were similar to the 4-12 µm diameter pores in the biofilm cross-section. The weight loss of the biofilms at 4 weeks was only 0.07%. The molecular weight of PCL biofilms did not change after immersion in phosphate buffered saline for 4 weeks. The failure loads of the biofilm were similar before (48 ± 9 N) and after immersion (47 ± 7 N, P > 0.1). The biofilm group had ~70% higher mean failure loads and 93% higher stiffness compared with the control group. CONCLUSIONS: We proposed and tested an internal fixation technique using a PCL biofilm to enhance tendon repair. Internal fixation with the biofilm followed by standard suturing can significantly increase the tensile strength of tendon repair sites. This technique has the potential to allow active motion rehabilitation during the early post-operative period.

[Prevalence and influence of diabetic retinopathy in populations at the age of ≥ 40 years in Luzhou City, Sichuan Province in 2011].

OBJECTIVE: To estimate the prevalence rate and risk factors of diabetic retinopathy(DR) in residents at the age of ≥ 40 years in Luzhou, Sichuan Province. METHODS: A multistage, stratified cluster random sampling method was used to select 7 communities of 8002 permanent residents ( ≥ 40 years old) for diabetes screened from May to December 2011 in Luzhou, Sichuan Province.Reference to the diagnostic criteria for diabetes recommended by World Health Organization in 1999, to the 1374 diabetes screening out patients with questionnaire, physical examination, fasting plasma glucose, 2-hours postprandial plasma glucose, glycosylated hemoglobin(HbAlc) and visual acuity, ocular diseases, fundus examination, etc.Some patients with fundus fluorescein angiography to the DR diagnosis, diagnostic criteria with reference to the diagnosis standard made by Chinese Ocular Fundus Diseases Society, Chinese Ophthalmological Society, Chinese Medical Association in 1984. Rates between the groups were compared using χ(2) test, trend χ(2) test or Fisher exact test, using multivariate analysis of risk factors associated with DR screening. RESULTS: A total of 8002 people were chosen and invited to take part in the project, 7478 persons completed the study, the overall response rate was 93.48%.One thousand three hundreds and seventy-four persons were diagnosed as diabetic patients, which took the proportion of 18.37% of the selected population, while 214 DR patients were found with the DR prevalence of 15.57%, occupying 2.86% of the total.Of 214 patients, 194 cases were non-proliferative diabetic retinopathy, making up 90.65% of DR patients, while 7 cases were found to be proliferative diabetic retinopathy, constitute 9.35% of DR patients. Multi-factor analysis manifest that there were close relationship between diabetic duration (trend χ(2) = 189.21, P < 0.01) , the level of HbAlc (χ(2) = 30.021, P < 0.01) , coronary heart disease (χ(2) = 4.075, P = 0.044) and DR prevalence. CONCLUSIONS: The prevalence of DR is high in the diabetic residents in Luzhou, Sichuan Province. Diabetic duration, the level of HbAlc and coronary heart disease are the significant factors to influence DR development. These results indicate that the changes of the level of HbAlc are closely related to the genesis and development of DR among the diabetic patients.

The lymphatic system: a therapeutic target for central nervous system disorders.

The lymphatic vasculature forms an organized network that covers the whole body and is involved in fluid homeostasis, metabolite clearance, and immune surveillance. The recent identification of functional lymphatic vessels in the meninges of the brain and the spinal cord has provided novel insights into neurophysiology. They emerge as major pathways for fluid exchange. The abundance of immune cells in lymphatic vessels and meninges also suggests that lymphatic vessels are actively involved in neuroimmunity. The lymphatic system, through its role in the clearance of neurotoxic proteins, autoimmune cell infiltration, and the transmission of pro-inflammatory signals, participates in the pathogenesis of a variety of neurological disorders, including neurodegenerative and neuroinflammatory diseases and traumatic injury. Vascular endothelial growth factor C is the master regulator of lymphangiogenesis, a process that is critical for the maintenance of central nervous system homeostasis. In this review, we summarize current knowledge and recent advances relating to the anatomical features and immunological functions of the lymphatic system of the central nervous system and highlight its potential as a therapeutic target for neurological disorders and central nervous system repair.

Metformin promotes angiogenesis and functional recovery in aged mice after spinal cord injury by adenosine monophosphate-activated protein kinase/endothelial nitric oxide synthase pathway.

Treatment with metformin can lead to the recovery of pleiotropic biological activities after spinal cord injury. However, its effect on spinal cord injury in aged mice remains unclear. Considering the essential role of angiogenesis during the regeneration process, we hypothesized that metformin activates the adenosine monophosphate-activated protein kinase/endothelial nitric oxide synthase pathway in endothelial cells, thereby promoting microvascular regeneration in aged mice after spinal cord injury. In this study, we established young and aged mouse models of contusive spinal cord injury using a modified Allen method. We found that aging hindered the recovery of neurological function and the formation of blood vessels in the spinal cord. Treatment with metformin promoted spinal cord microvascular endothelial cell migration and blood vessel formation in vitro. Furthermore, intraperitoneal injection of metformin in an in vivo model promoted endothelial cell proliferation and increased the density of new blood vessels in the spinal cord, thereby improving neurological function. The role of metformin was reversed by compound C, an adenosine monophosphate-activated protein kinase inhibitor, both in vivo and in vitro, suggesting that the adenosine monophosphate-activated protein kinase/endothelial nitric oxide synthase pathway likely regulates metformin-mediated angiogenesis after spinal cord injury. These findings suggest that metformin promotes vascular regeneration in the injured spinal cord by activating the adenosine monophosphate-activated protein kinase/endothelial nitric oxide synthase pathway, thereby improving the neurological function of aged mice after spinal cord injury.

NeuroD1 A45T and PAX4 R121W polymorphisms are associated with plasma glucose level of repaglinide monotherapy in Chinese patients with type 2 diabetes.

AIMS: We aimed to determine whether NeuroD1/BETA2 and PAX4 polymorphisms were associated with the therapeutic efficacy of repaglinide in Chinese type 2 diabetes mellitus (T2DM) patients. METHODS: Three hundred and sixty-eight T2DM patients and 132 healthy control subjects were genotyped by restriction fragment length polymorphism. Forty-three patients with various genotypes were randomly selected to undergo 8 weeks of repaglinide treatment (3 mg day(-1)). Fasting plasma glucose, postprandial plasma glucose, glycated haemoglobin, fasting and postprandial serum insulin (FINS, PINS), homeostasis model assessment for insulin resistance, serum triglyceride, total cholesterol, low-density lipoprotein-cholesterol and high-density lipoprotein-cholesterol were determined before and after repaglinide treatment. RESULTS: The allelic frequency of NeuroD1/BETA2 T45 was higher in T2DM patients than in the control subjects [13.45 vs. 6.82%, P < 0.01, odds ratios = 2.342 (1.365, 4.019), P= 0.002]. Type 2 diabetes mellitus patients with the mutated allele of NeuroD1/BETA2 A45T polymorphism showed higher FINS (13.46 ± 12.57 vs. 10.04 ± 7.09 mU l(-1) , P < 0.05) (11.67, 14.83 vs. 8.38, 11.37) and PINS (52.11 ± 40.93 vs. 68.66 ± 43.87 mU l(-1), P < 0.05) (44.89, 58.35 vs. 55.35, 88.87) than individuals with the T allele. The PAX4 R121W R allele carriers had higher PINS (52.11 ± 40.93 vs. 68.66 ± 43.87 mU l(-1), P < 0.05) (44.89, 58.35 vs. 55.35, 88.87) than subjects with the W allele. After repaglinide treatment, patients with the T allele of NeuroD1/BETA2 A45T polymorphisms had attenuated efficacy on fasting plasma glucose (-2.79 ± 2.14 vs.-0.99 ± 1.80 mmol l(-1), P < 0.01) (-3.53, -1.84 vs.-1.99, -0.13) and postprandial plasma glucose (-6.71 ± 5.90 vs.-2.54 ± 3.39 mmol l(-1), P < 0.01) (-9.28, -4.62 vs.-4.34, -0.84). Patients with the RR genotype of PAX4 R121W showed better efficacy with respect to the level of postprandial plasma glucose than R/W genotypes (-6.53 ± 6.52 vs.-2.95 ± 1.17 mmol l(-1), P < 0.05) (-8.20, -4.89 vs.-3.92, -1.20). CONCLUSIONS: The NeuroD1/BETA2 and PAX4 polymorphisms were substantially associated with plasma glucose level after repaglinide monotherapy.

[Study on the differentially expressed microRNA of human retinal microvascular endothelial cells in high glucose environment by microRNA gene chip].

OBJECTIVES: To explore the differentially expressed microRNA (miRNA) of human retinal microvascular endothelial cell (HRCEC) in hyperglycemic environment by miRNA gene chip, then adopt bioinformatics methods to forecast target genes of part differentially expressed miRNA. METHODS: Experimental study. HRCEC were cultured. Took the 3-4 generation growth good cells and divided the cells into three groups: (1) normal control group: DMEM medium with 25 mmol/L glucose; (2) high glucose group: conditioned medium with 90 mmol/L glucose; (3) mannitol high permeability control group: conditioned medium with 65 mmol/L mannitol and 25 mmol/L glucose. Each group cells were cultured in the above conditions for five days, then used in situ cell death detection kit for apoptosis detection; the total RNA was isolated and examined; the differentially expressed miRNA were detected by miRNA gene chip, part results of miRNA array were verified by real-time quantitate polymerase chain reaction (PCR), potential miRNA targets were analyzed by bioinformatics methods. RESULTS: Observed apoptotic HRCEC by fluorescence microscope: the nucleus of normal control group and mannitol control group were dyed by DAPI and appeared blue fluorescence, but hadn't apoptosis fluorescent signals; the nucleus of high glucose group also appeared blue fluorescence, and had green apoptosis fluorescent signals. Quality testing of total RNA: with spectrophotometer measurement, the ratio of absorbance of total RNA in normal control group at A(260)/A(280) nm was 1.99, at A(260)/A(230) was 2.05;total RNA of high glucose group at A(260)/A(280) was 1.98, at A(260)/A(230) was 2.26. The results of formaldehyde degeneration agarose gel electrophoresis showed that the electrophoresis strips were clear and complete, indicated that the total RNA had better quality and high purity. Compared with normal control group, 49 miRNAs were found to be differentially expressed in high glucose group (fold change > 2 and fold change < 0.5), including 31 up-regulated miRNAs and 18 down-regulated miRNAs. The results of real-time quantity PCR revealed that hsa-miR-320c and hsa-miR-29a(*) were up-regulated in high glucose group, which were consistence with the miRNA gene chip. Furthermore, the target genes prediction of two above miRNAs were involved many growth factors and proteins. CONCLUSION: miRNA are differently expressed in HRCEC under hyperglycemic conditions.

[CCR1 expression on immune cells in orbital tissue of patients with thyroid associated ophthalmopathy].

OBJECTIVE: To investigate the expression of CC chemokine receptor 1(CCR1) on the immune cells in orbital tissue of patients with thyroid associated ophthalmopathy(TAO). METHODS: The tissue samples of orbital tissue were collected from 25 cases of TAO patient and 10 normal control subjects, and the expression of CCR1 in the immune cells was studied by SP immunohistochemistry. The correlation between CCR1 and clinical activity scores (GAS) was analyzed. RESULTS: The expression of CCR1 on immune cells was positive, in orbital tissue of patients with TAO, and the positive rate was 84%, while it was negative or low positive in normal control subjects. The difference between them was significant (P < 0.05). In addition, the expression of CCR1 on the immune cells of orbital tissue with TAO had significant correlation with CAS (P < 0.05). CONCLUSION: The expression of chemokine receptor CCR1 on the immune cells in orbital tissue of TAO patients is higher than that of normal subjects. CCR1 may play a role in the pathogenesis of TAO.

[Distribution Characteristics and Ecological and Health Risk Assessment of Phthalic Acid Esters in Surface Water of Qiandao Lake, China].

In order to reveal the pollution and risk level of phthalic acid esters (PAEs) in Qiandao Lake, six types of PAEs in 17 sampling points (in Qiandao Lake and its inflowing rivers) in dry and wet seasons were detected. The results showed that six types of PAEs were detected in both dry and wet seasons, with the concentrations of 0.98-5.33 µg·L-1 (average concentration 2.63 µg·L-1) in the dry season and 3.22-17.88 µg·L-1 (average concentration 7.99 µg·L-1) in the wet season. In terms of the detection rate and concentration of each monomer PAEs, DiBP, DBP, and DEHP were the main PAEs components in the water body. The measured value of DBP at 10 sampling points and its average mass concentration in the wet season were higher than the national standard (3 µg·L-1). Principal component analysis indicated that the main sources of PAEs were personal care products, plastics, and domestic waste. The pollution level of PAEs in Qiandao Lake was at a high level at home and abroad. The health risk assessment results in Qiandao Lake showed that the non-carcinogenic risk index of PAEs in the study area was less than 1, which would not produce non-carcinogenic risks to the human body. The carcinogenic risk index of children exceeded 10-6 at some points, indicating that it may pose carcinogenic risks to children, to which more attention should be paid.

[Analysis of Heavy Metal Pollution Characteristics and Potential Ecological Risks of Surface Sediments in Dongjiang Lake].

To understand the pollution characteristics, spatial distribution characteristics, potential ecological risks, and sources of heavy metals in surface sediments of Dongjiang Lake, 12 surface sediment samples were collected from Dongjiang Lake. The contents of 20 heavy metals including Li, Be, V, Cr, Mn, Co, Ni, Cu, Zn, Ga, As, Se, Rb, Sr, Ag, Cd, Cs, Ba, Pb, and U were analyzed in this study. The geo-accumulation index method and potential risk index method were used to evaluate the pollution degree and potential risks of heavy metals in sediments, and the correlation analysis and principal component analysis were used to trace the source of the primary heavy metals. The results showed that the average contents of ω(Cd)(2.25 mg·kg-1) and ω(As)(80.80 mg·kg-1) in heavy metals of Dongjiang Lake sediments were 21.2 times and 5.5 times the background value (0.11 mg·kg-1 and 14.7 mg·kg-1) of Hunan province. The overall spatial distribution of heavy metals was in the order of South>North>Central. The evaluation by the geo-accumulation index method showed that Cd was at the heavy pollution level, As and Se were at the moderate pollution level, Ag and Ga were at the light pollution level, and the other heavy metals were below the pollution standard. The evaluation by the potential risk index showed that all the surface sediments of Dongjiang Lake were at the high-risk level. The main environmental risk factor was Cd, which had an extremely high risk; the second was As, which had a medium risk; and the remaining heavy metals had no ecological risk.

Serine racemase rs391300 G/A polymorphism influences the therapeutic efficacy of metformin in Chinese patients with diabetes mellitus type 2.

1. The aim of this study was to investigate the association of the serine racemase (SRR) rs391300 G/A polymorphism with the risk of diabetes mellitus type 2 (T2DM) and to assess the impacts of the polymorphism on the therapeutic efficacy of metformin in Chinese patients. 2. A case-control study of 402 patients with T2DM and 171 healthy controls was conducted. The SRR rs391300 polymorphism was genotyped in all participants using the ABI 3700 automated sequencer. Forty-four recent-onset T2DM patients with different rs391300 genotypes were selected to receive 500 mg metformin orally daily for 12 consecutive weeks as monotherapy. Serum fasting plasma glucose (FPG), postprandial plasma glucose (PPG), glycated haemoglobin (HbA1c), fasting serum insulin (FINS), postprandial serum insulin (PINS), triglycerol (TG), cholesterol (CHO), low-density lipoprotein cholesterol (LDL-c), high-density lipoprotein cholesterol (HDL-c), homeostasis model assessment for insulin resistance (HOMA-IR), and body mass index (BMI) were determined before and after metformin treatment. 3. The distribution frequencies of rs391300 were in agreement with Hardy-Weinberg equilibrium (P > 0.05). After treatment with metformin, the values of BMI, FPG, PPG, PINS, HbA1c, CHO, and TG decreased significantly (P < 0.01), whereas FINS increased (P < 0.001), in patients with T2DM. Patients with the GA or AA genotype of rs391300 showed better improvements in the levels of FPG, PPG, and CHO (P < 0.05) than individuals with the GG genotype. 4. The SRR rs391300 polymorphism was associated with the therapeutic efficacy of metformin in Chinese patients with T2DM.

NAMPT -3186C/T polymorphism affects repaglinide response in Chinese patients with Type 2 diabetes mellitus.

1. In the present study, we investigated the associations of nicotinamide phosphoribosyltransferase (NAMPT)-3186 C/T and -948G/T polymorphisms with the risk of Type 2 diabetes mellitus (T2DM) and their impact on the efficacy of repaglinide in Chinese Han T2DM patients. 2. In all, 170 patients with T2DM and 129 healthy controls were genotyped for NAMPT-948G>T and -3186C>T polymorphisms. Thirty-five patients with different NAMPT -3186 C/T genotypes and the same organic anion-transporting polypeptide 1B1 (OATP1B1521) T/C genotype were randomly selected to undergo 8 weeks preprandial repaglinide treatment (1 mg, three times daily). Serum fasting plasma glucose (FPG), post-prandial plasma glucose (PPG), glycated haemoglobin (HbAlc), fasting serum insulin (FINS), post-prandial serum insulin (PINS), triglyceride (TG), total cholesterol (CHO), homeostasis model assessment of insulin resistance (HOMA-IR), low-density lipoprotein-cholesterol (LDL-C) and high-density lipoprotein-cholesterol (HDL-C) were determined before and after repaglinide treatment. 3. After repaglinide treatment for 8 consecutive weeks, there were significantly decreases in PFG, PPG, HbAlc, CHO and LDL-C, and increases in FINS, HDL-C and the HDL-C : LDL-C ratio, in T2DM patients. The elevated PINS value in patients with CT genotypes was significantly lower than that in patients with the CC and TT genotypes (P < 0.05) and there were significant differences in CHO between patients with the CT genotype and the CC or TT genotype (P < 0.05). 4. The data suggest that the NAMPT -3186C>T polymorphism is significantly associated with plasma levels of PINS and CHO in Chinese T2DM patients with repaglinide monotherapy.

Utx Regulates the NF-κB Signaling Pathway of Natural Stem Cells to Modulate Macrophage Migration during Spinal Cord Injury.

Neural stem cells (NSCs) play vital roles in the homeostasis of neurological function. Ubiquitously transcribed tetratricopeptide repeat, X chromosome (UTX) is an important regulator of stem cell phenotypes. In our current study, we aimed to investigate whether the conditional knockout of UTX on neural stem cells alters macrophage assembly in response to spinal cord injury (SCI). Conditional knockout Utx of NSC (Utx-KO) mice was used to generate SCI models by the modified Allen method. We reported that neurological function and scar hyperplasia significantly improved in Utx-KO mice after SCI, accompanied by significantly reduced assembly of macrophages. With a 45-fold pathway array and Western blot, we found that Utx-KO could significantly inhibit NF-κB signaling activation and promote the synthesis and secretion of macrophage migration inhibitory factor (MIF) in NSCs. Administration of the selective NF-κB p65 activator betulinic acid and the selective MIF inhibitor ISO-1 confirmed that the activation of NF-κB p65 phosphorylation or inhibition of MIF could eliminate the benefits of Utx-KO in SCI, such as inhibition of macrophage aggregation and reduction in scar proliferation. This study confirmed that UTX in NSCs could alter macrophage migration and improve neurological function recovery after SCI in mice.

Silencing of lncRNA PKIA-AS1 Attenuates Spinal Nerve Ligation-Induced Neuropathic Pain Through Epigenetic Downregulation of CDK6 Expression.

Neuropathic pain (NP) is among the most intractable comorbidities of spinal cord injury. Dysregulation of non-coding RNAs has also been implicated in the development of neuropathic pain. Here, we identified a novel lncRNA, PKIA-AS1, by using lncRNA array analysis in spinal cord tissue of spinal nerve ligation (SNL) model rats, and investigated the role of PKIA-AS1 in SNL-mediated neuropathic pain. We observed that PKIA-AS1 was significantly upregulated in SNL model rats and that PKIA-AS1 knockdown attenuated neuropathic pain progression. Alternatively, overexpression of PKIA-AS1 was sufficient to induce neuropathic pain-like symptoms in uninjured rats. We also found that PKIA-AS1 mediated SNL-induced neuropathic pain by directly regulating the expression and function of CDK6, which is essential for the initiation and maintenance of neuroinflammation and neuropathic pain. Therefore, our study identifies PKIA-AS1 as a novel therapeutic target for neuroinflammation related neuropathic pain.

Real-time ultrasonic assessment of progressive proteoglycan depletion in articular cartilage.

The loss of proteoglycan (PG) is regarded as one of the early signs of osteoarthritis (OA), thus observing the progress of PG loss would be useful for the early detection of OA. In this study, high-frequency ultrasound was used to monitor and analyze the trypsin-induced progressive degeneration in articular cartilage. Full thickness cartilage-bone specimens (n = 10) prepared from normal bovine patellae were digested using 0.25% trypsin solution for different periods of time to evaluate the dynamics of the digestion process. The trypsin penetration front was observed in M-mode image, which was acquired using a nominal 50 MHz focused transducer. The transient speed of the digestion process was estimated from the image. The digestion fraction, which represents the ratio of the digestion depth to the total cartilage thickness, was estimated from ultrasound data and histology sections. With ultrasound, the digestion fraction observed in the 10 specimens ranged from 64% to 99% and was correlated to that measured by histology (R(2) > or = 0.63, p < 0.05). It was found that the digestion speed decreased nonlinearly with depth from 0.61 +/- 0.16 microm/s (mean +/- SD) in the superficial zone to 0.04 +/- 0.02 microm/s in a region located at 70% of the cartilage thickness in depth. The relationship between the digestion depth and the exposure duration in trypsin could be described using a third order polynomial function. The full thickness of digested and undigested tissues was also measured using caliper, estimated from ultrasound data and histology sections, and compared. These findings indicate that ultrasound could provide useful information about the trypsin-induced progressive PG depletion in articular cartilage. Therefore, ultrasound represents a useful tool to evaluate the dynamics of models of OA in vitro in cartilage specimens in a research environment and this would ultimately help the in vitro examination of articular cartilage for research related to model of OA from the early stages of tissue degradation.

[Tetramethylpyrazine accelerated spinal cord repair through regulation of caspase-3 and neurofilament protein expression: an acute spinal cord injury model in rats].

OBJECTIVE: To investigate the effect of tetramethylpyrazine on spinal cord repair through hypothesized pathways including regulation of caspase-3, neurofilament protein NF-L, NF-H, and NF-M expression. METHODS: Allenos weight drop method was used to establish acute spinal cord injury (SCI) rat model at T10 section and was conducted in 80 adult Sprague Dawley rats that were subsequently divided into pseudo-therapy group (sodium chloride group, n=40) and tetramethylpyrazine group (treatment group, n=40). Eight normal rats were included into the pseudo-surgery normal group (n=1 for each time point). The function of spinal cord was evaluated with animal behavioral scores by measuring modified Rivilin loxotic plate degree and counting BBB score at 1, 3, 7, 14, and 21 d postoperatively. The injured spinal cord tissue samples were harvested at 1 h, 3 h, 6 h, 1 d, 3 d, 7 d, 14 d, and 21 d postoperatively (n=5 for each time point) for the preparation of continuous histological sections that were analyzed the expression of caspase-3,NF-L,NF-H, and NF-M by immunohistochemistry method. RESULTS: At postoperative Day 7, 14, and 21, animal behavioral scores revealed higher modified Rivlin loxotic plate degree and BBB scores in the treatment group when compared with those of the control group (P<0.05). The cells in the injured spinal cord tissue expressed significantly less caspase-3 and significantly more NF (NF-L, NF-H and NF-M) in the treatment group than those in the control group at Day 3, 7, and 14 (P<0.05). There was positive correlation between modified Rivlin loxotic plate degree and NF expression, BBB score and NF expression; and negative correlations between BBB score and caspase-3 expression, caspase-3 expression and NF expression. CONCLUSION: Tetramethylpyrazine improves spinal cord healing through regulation of caspase-3 and neurofilament protein expression.

[Effect of low-intensity pulsed ultrasound on the enchondral bone formation in posterolateral lumbar fusion in rabbits].

OBJECTIVE: To observe the effect of daily low-intensity pulsed ultrasound (LIPUs) therapy on improving the enchondral bone formation in lumbar fusion in rabbit models, and to explore its possible mechanism. METHODS: Posterolateral noninstrumented bilateral fusions were performed at the L5 approximately L6 levels in 20 New Zealand rabbits. The autograft iliac bone was implanted on the left side, and the hydroxyapatite bioceramic artificial bone on the right. The rabbits were divided into a treatment group and a control group randomly. One week after the surgery, LIUPs was administered for 20 minutes per day for 4 weeks over the fusion site in the treatment group and false treatment was used in the control group. Post-anterior X-ray photographs were taken to determine the conditions of fusion area, and then, rabbits were killed and the fusion tissues were obtained. Chondrocytes were detected by histological and cytological methods. RESULTS: Compared with the control group, the fusion rate of the treatment group was significantly up-regulated (P<0.05). There was plenty bone trabecula in the fusion area in the treatment group, the number of chondrocytes was also higher than that of the control group (P<0.05), and there was no statistical difference in the number of chondrocytes between the iliac and artificial bone tissues after the treatment(P>0.05). CONCLUSION: Low-intensity pulsed ultrasound therapy improves the endochondral bone formation in the lumbar spine fusion in rabbit models.

Synchrotron radiation micro-CT as a novel tool to evaluate the effect of agomir-210 in a rat spinal cord injury model.

MicroRNA-210 (miR-210) was initially reported to be associated with hypoxia and plays a vital role in modulating angiogenesis. However, the potential effect and underlying mechanisms of miR-210 activity in rat spinal cord injury (SCI) have not yet been fully illuminated. In the present study, differential microRNA expression after SCI was determined by Microarray analysis. To explore the effect of miR-210 after SCI, we intrathecally injected agomir-210 with Alzet Osmotic Pumps to up-regulated the endogenous miR-210 expression. Then, synchrotron radiation micro-CT (SRµCT) imaging was used to investigate the effect of agomir-210 in rat SCI model. We found that the endogenous miR-210 expression could be up-regulated by intrathecal agomir-210 injection. The administration of agomir-210 significantly promoted angiogenesis, as evidenced by increased vessel number and volume detected by SRµCT, attenuated the lesion size and improved functional recovery after SCI. Additionally, agomir-210 attenuated cellular apoptosis and inflammation in the injured rat spinal cord. Expression levels of pro-apoptotic protein (Bax) and pro-inflammatory cytokines (TNF-α and IL-1ß) were significantly decreased after agomir-210 treatment, whereas expression levels of anti-apoptotic (Bcl-2) and anti-inflammatory (IL-10) proteins were up-regulated. In conclusion, our results indicated that SRµCT is a powerful imaging tool to evaluate the effects of angiogenesis after agomir-210 administration in rat SCI model. The up-regulation of endogenous miR-210 expression following agomir-210 administration promoted angiogenesis and anti-apoptotic protein expression, and attenuated inflammation. MiR-210 played a positive role in neurological functional recovery and could be a potential new therapeutic target for SCI.

Tetramethylpyrazine Facilitates Functional Recovery after Spinal Cord Injury by Inhibiting MMP2, MMP9, and Vascular Endothelial Cell Apoptosis.

BACKGROUND: Spinal cord injury (SCI) is a major public health issue that leads to neurological dysfunctions and morbidities in patients. Tetramethylpyrazine (TMP) plays a neuroprotective role in SCI; however, the underlying mechanism has not been fully elucidated. OBJECTIVE: In the present study, we aimed to investigate the mechanisms and therapeutic effects of TMP on SCI. METHODS: A contusion SCI model was established that used a modified Allen's method. In the TMP group, TMP (200 mg/kg) was injected daily for 5 days post-injury, while in the Negative Control (NC) group, an equal volume of normal saline was injected. Hindlimb motor function was evaluated using the Basso, Beattie, Bresnahan (BBB) scale. The effects of TMP on protein levels of the matrix metalloproteinases 2 (MMP2) and 9 (MMP9), Bax and cleaved caspase-3 were determined by western blotting. Apoptotic changes in vascular endothelial cells were evaluated using immunofluorescence and TUNEL staining. Alterations in 3D vessel morphology after treatment with TMP were assessed by synchrotron radiation micro-CT (SRµCT). RESULTS: TMP treatment significantly improved recovery in hindlimb motor function and attenuated vascular endothelial cell apoptosis in rats with SCI. Additionally, TMP treatment markedly decreased the protein levels of MMP2 and MMP9, pro-apoptotic bax and cleaved caspase-3 while promoting angiogenesis, as evidenced by vessel visualization using SRµCT. CONCLUSION: These results indicate that TMP attenuated SCI-induced neurological impairments by the down-regulation of the expression of MMP2 and MMP9 proteins, the inhibition of vascular endothelial cell apoptosis, and the promotion of angiogenesis.