AIM: To provide references for the non-clinical evaluation of therapeutic targets or drugs for retinoblastoma, fluorescently labeled Y79 cells are injected into the vitreous body of BALB/c-nu mice to establish a retinoblastoma model, and the Melphalan treatment group is used as a positive control, which is verified by fluorescence imaging technology.METHODS: BALB/c-nu mice were intravitreous injected with GFP transfected Y79 cells(1.0×107 cell/mL, 3 μL)to establish the model. On the 27th day, the mice were randomly divided into model control group and different doses of Melphalan groups(1, 3, 10 μg/eye groups)according to the fluorescence value of in vivo imaging, with vitreous body single administrated and ocular symptoms observed daily. Slit-lamp examination was performed at 12, 20, 29, 35, 42, 48, 55, 76, and 83 d after modeling. In vivo imaging was performed on 12, 20, 27, 41, 48, 55, 62, 69, 76, and 83 d. At the last treatment, the eyeball, brain and cerebellum tissues were removed for histopathological examination.RESULTS: From the sixth day of modeling, cloud-like substances could be seen in the eyes of the animals, and the cloud-like substances occupied the whole eyeball of the mice in the model control group at the later stage, accompanied by irregular growth of blood vessels. After 27 days of modeling, the fluorescence value was detected in all the animals, and the fluorescence value continued to increase with the extension of modeling time. The fluorescence value of the tumor reached the peak after 69-83 days of modeling. Histological examination showed severe proliferation of intraocular tumor cells in the model control group, and tumor cells were observed in the brain of 1 model animal. In the 10 μg/eye Melphalan group, the fluorescence value was significantly decreased at 17 d after administration. The fluorescence value of the 3 μg/eye Melphalan group was significantly inhibited at 59 d after administration. No tumor cells were found in the brain tissue of animals in all Melphalan groups.CONCLUSION: After vitreous injection of Y79/pCDH-LUC-copGFP cells in BALB/c-nu mice, significant ocular lesions and proliferation of tumor cells were observed in the eyes. Meanwhile, Melphalan intervention significantly inhibited tumor cells in a dose-dependent manner, indicating that the mouse model of retinoblastoma was successfully constructed.

Objective To systematically investigate the effects of Dangmu extract syrup on the growth and development of 4-day-old(postnatal day 4,PND4)Sprague-Dawley(SD)rats and its toxic reactions.Methods According to the whole litter design method,128 young mice(PND2)were randomly divided into negative control group and low,medium and high dose groups.From PND4,the animals were orally given pure water,31 g/kg,93 g/kg and 280 g/kg(calculated as raw herb material)of Dangmu extract syrup,respectively,once daily for 18 consecutive days,with a 15 d of recovery phase.During the study period,the general state,growth and development,nerve reflex function,spontaneous behavior,hematology,coagulation,blood biochemistry,immune function,growth hormone and histopathology of the animals in each group were observed or examined.Results After 18 d of continuous administration,compared with the negative control group,GLU(male and female)in the medium and high dose groups increased(P＜0.05 or P＜0.01),LDH(male and female)and AST(male)in the medium and high dose groups,ALT,AST(female)in the high dose group decreased(P＜0.05 or P＜0.01),RET and percentage of RET(male and female)in the low and high dose groups,RET(male)in the medium dose group increased(P＜0.05 or P＜0.01),spleen mass and the organ-to-body mass ratio(male and female)in the low and high dose groups and female in the medium dose group increased(P＜0.05 or P＜0.01).Splenic nodule structures were formed in all dose groups with large size and number,and there was a dose relationship in the degree of changes.After 15 d recovery period,compared with the negative control group,GLU(female)in the low dose group increased(P＜0.05),ALT,AST,ALP,TG(female)in the medium and high dose groups,GGT,TG,TCHO(male)in the medium dose groups,AST,ALP,TG,LDH(male)in the high dose group decreased(P＜0.05 or P＜0.01),RET(female)and percentage of RET(male)in the high dose group increased(P＜0.05).compared with the 18 d of continuous administration,the spleen structures of the animals in each group were more completely developed and the splenic nodule structures were obvious,but no significant difference was noted in the comparison between groups.No significant drug-related changes were observed in other test result.Conclusions Dangmu extract syrup advanced the development of complete spleen structure in 4-day-old SD rats,accompanied by the enhancement of its hematopoietic function,and at the same time,it caused the animals,blood glucose to rise,the enhancement of glucose metabolism function led to the increase of related enzyme consumption,and led to the decrease of some liver function parameters,and showed a dose correlation.There was no gender difference in the changes,which were reversible after stop administration,and the mechanism of the changes needs to be further explored and confirmed.In the clinical trials,attention should be paid to the control of the dose of the test article,and regular monitoring of the spleen and related blood and clinical chemistry parameters.

Objective To establish a dexamethasone(Dex)-induced zebrafish model of glucocorticoid-induced osteoporosis(GIOP)combined with glucocorticoid-induced hypertension(GIHT),and to validate the model by the systematic evaluation of both the phenotypic manifestations and molecular mechanisms.Methods Zebrafish larvae at 3 or 4 d post-fertilization(dpf)were divided randomly into a control group(0.1％dimethyl sulfoxide)and a model group(10 μmol/L Dex).Osteogenic parameters and vessel diameter were assessed at 0,48,and 96 h post-administration(n=10).Bone mineralization and density were determined by the total area and sum brightness after Alizarin red staining.Vessel diameter was measured by detecting blood flow in the dorsal aorta.After confirming the optimal administration time,expression levels of bone-formation-related proteins(protein kinase B(Akt),glycogen synthase kinase(GSK)-3β,β-catenin)and angiogenesis-related proteins(AMP-activated protein kinase(AMPK),nuclear factor(NF)-κB)were detected by Western Blot to verify the molecular effectiveness of the model.Results Exposure to Dex for 96 h reduced bone mineralization and density in zebrafish larvae compared with the control group,and statistical analysis identified 4 dpf zebrafish and Dex administration for 96 h as the optimal modeling times for the GIOP model.Blood vessel diameter was significantly decreased in the model group compared with the control group(P＜0.05),and the difference became more pronounced with longer administration time and was particularly evident at 4 dpf and treatment for 96 h.Western Blot analysis showed that Dex significantly decreased protein expression levels of Akt,β-catenin,and NF-κB(P＜0.05)and significantly increased the expression of GSK-3β and AMPK(P＜0.05),suggesting that Dex effectively inhibited bone formation and angiogenesis after 96 hours treatment in 4 dpf zebrafish.Conclusions Treatment of 4 dpf zebrafish larvae with 10 μmol/L Dex rapidly established a reliable zebrafish model of GIOP combined with GIHT,providing an ideal animal model for further studies of the common mechanisms of the two diseases and for screening new drugs.

Objective To investigate the effects of the polymer pharmaceutical excipient methoxy poly-ethylene glycol poly-lactic acid(mPEG-PLA)in Sprague-Dawley(SD)rats and Beagle dogs and its toxicological reactions,to provide a reference for its safe clinical use.Methods SD rats and Beagle dogs(male∶female ratios,1∶1)were divided randomly into control group and low,medium,and high dose mPEG-PLA groups(70,210,700 mg/kg).Animals received intravenous mPEG-PLA once a day for 90 days,followed by a 28-day recovery period.Indicators including clinical observations,food intake,body weight,hematology,blood biochemistry,immune function,and pathological examination were recorded.Results Compared with the control group,(1)food intake was decreased(P＜0.01)and body weight was increased(P＜0.05 or P＜0.01)after 90 days of continuous administration,with similar changes in the medium and high dose groups in both rats and dogs.In addition,MONO/MONO％,RBC,MCH,MCHC,HCT,HGB,PLT,TP,ALB,GLB,and Fbg were all decreased(P＜0.05 or P＜0.01)and coagulation indexes(e.g.,APTT)were increased(P＜0.05 or P＜0.01).Organ weights and the organ-to-body/brain weight ratios of the liver and spleen were increased(P＜0.05 or P＜0.01),and histopathology indicated numerous foam-like macrophages in the hepatic sinuses,red spleen pulp,and lymph node medulla.DBIL and TBIL also increased in rats in the high dose group(P＜0.05 or P＜0.01),while the dogs experienced skin swelling or scabs,abdominal swelling,vomiting,decreased activity,high albuminuria,and ascites,and the renal glomerular cells showed vacuoles.(2)After 28 days of recovery,rats and dogs in the medium and high dose groups showed a few foam-like macrophages in the hepatic sinuses,red spleen pulp,and lymph node medulla,as well as decreased of food intake in dogs.The MCHC,PLT,and TP decreased in dogs in the high dose group(P＜0.05),and the liver and spleen weights and organ coefficients in rats increased(P＜0.05 or P＜0.01),while the MONO％decreased in male rats in the medium dose group(P＜0.05).Conclusions Administration of mPEG-PLA 210 and 700 mg/kg for 90 days caused blood mononuclear cells to enter and aggregate in the liver,spleen,lymph nodes,and other tissues in SD rats and Beagle dogs,leading to secondary tissue structural damage.Protein and fibrinogen synthesis and bilirubin metabolism in the liver decreased,leading to abnormal coagulation function,and decreased intravascular colloid osmotic pressure resulted in edema and bleeding.The result suggest that the liver,spleen,kidney,and lymph nodes are target organs for mPEG-PLA toxicity,with dose-dependent and reversible effects and species differences,but no significant sex differences.Clinical monitoring of related organ functions is needed to avoid secondary damage.

Objective To establish a dexamethasone(Dex)-induced zebrafish model of glucocorticoid-induced osteoporosis(GIOP)combined with glucocorticoid-induced hypertension(GIHT),and to validate the model by the systematic evaluation of both the phenotypic manifestations and molecular mechanisms.Methods Zebrafish larvae at 3 or 4 d post-fertilization(dpf)were divided randomly into a control group(0.1％dimethyl sulfoxide)and a model group(10 μmol/L Dex).Osteogenic parameters and vessel diameter were assessed at 0,48,and 96 h post-administration(n=10).Bone mineralization and density were determined by the total area and sum brightness after Alizarin red staining.Vessel diameter was measured by detecting blood flow in the dorsal aorta.After confirming the optimal administration time,expression levels of bone-formation-related proteins(protein kinase B(Akt),glycogen synthase kinase(GSK)-3β,β-catenin)and angiogenesis-related proteins(AMP-activated protein kinase(AMPK),nuclear factor(NF)-κB)were detected by Western Blot to verify the molecular effectiveness of the model.Results Exposure to Dex for 96 h reduced bone mineralization and density in zebrafish larvae compared with the control group,and statistical analysis identified 4 dpf zebrafish and Dex administration for 96 h as the optimal modeling times for the GIOP model.Blood vessel diameter was significantly decreased in the model group compared with the control group(P＜0.05),and the difference became more pronounced with longer administration time and was particularly evident at 4 dpf and treatment for 96 h.Western Blot analysis showed that Dex significantly decreased protein expression levels of Akt,β-catenin,and NF-κB(P＜0.05)and significantly increased the expression of GSK-3β and AMPK(P＜0.05),suggesting that Dex effectively inhibited bone formation and angiogenesis after 96 hours treatment in 4 dpf zebrafish.Conclusions Treatment of 4 dpf zebrafish larvae with 10 μmol/L Dex rapidly established a reliable zebrafish model of GIOP combined with GIHT,providing an ideal animal model for further studies of the common mechanisms of the two diseases and for screening new drugs.

Objective To systematically investigate the effects of Dangmu extract syrup on the growth and development of 4-day-old(postnatal day 4,PND4)Sprague-Dawley(SD)rats and its toxic reactions.Methods According to the whole litter design method,128 young mice(PND2)were randomly divided into negative control group and low,medium and high dose groups.From PND4,the animals were orally given pure water,31 g/kg,93 g/kg and 280 g/kg(calculated as raw herb material)of Dangmu extract syrup,respectively,once daily for 18 consecutive days,with a 15 d of recovery phase.During the study period,the general state,growth and development,nerve reflex function,spontaneous behavior,hematology,coagulation,blood biochemistry,immune function,growth hormone and histopathology of the animals in each group were observed or examined.Results After 18 d of continuous administration,compared with the negative control group,GLU(male and female)in the medium and high dose groups increased(P＜0.05 or P＜0.01),LDH(male and female)and AST(male)in the medium and high dose groups,ALT,AST(female)in the high dose group decreased(P＜0.05 or P＜0.01),RET and percentage of RET(male and female)in the low and high dose groups,RET(male)in the medium dose group increased(P＜0.05 or P＜0.01),spleen mass and the organ-to-body mass ratio(male and female)in the low and high dose groups and female in the medium dose group increased(P＜0.05 or P＜0.01).Splenic nodule structures were formed in all dose groups with large size and number,and there was a dose relationship in the degree of changes.After 15 d recovery period,compared with the negative control group,GLU(female)in the low dose group increased(P＜0.05),ALT,AST,ALP,TG(female)in the medium and high dose groups,GGT,TG,TCHO(male)in the medium dose groups,AST,ALP,TG,LDH(male)in the high dose group decreased(P＜0.05 or P＜0.01),RET(female)and percentage of RET(male)in the high dose group increased(P＜0.05).compared with the 18 d of continuous administration,the spleen structures of the animals in each group were more completely developed and the splenic nodule structures were obvious,but no significant difference was noted in the comparison between groups.No significant drug-related changes were observed in other test result.Conclusions Dangmu extract syrup advanced the development of complete spleen structure in 4-day-old SD rats,accompanied by the enhancement of its hematopoietic function,and at the same time,it caused the animals,blood glucose to rise,the enhancement of glucose metabolism function led to the increase of related enzyme consumption,and led to the decrease of some liver function parameters,and showed a dose correlation.There was no gender difference in the changes,which were reversible after stop administration,and the mechanism of the changes needs to be further explored and confirmed.In the clinical trials,attention should be paid to the control of the dose of the test article,and regular monitoring of the spleen and related blood and clinical chemistry parameters.

Objective To investigate the effects of the polymer pharmaceutical excipient methoxy poly-ethylene glycol poly-lactic acid(mPEG-PLA)in Sprague-Dawley(SD)rats and Beagle dogs and its toxicological reactions,to provide a reference for its safe clinical use.Methods SD rats and Beagle dogs(male∶female ratios,1∶1)were divided randomly into control group and low,medium,and high dose mPEG-PLA groups(70,210,700 mg/kg).Animals received intravenous mPEG-PLA once a day for 90 days,followed by a 28-day recovery period.Indicators including clinical observations,food intake,body weight,hematology,blood biochemistry,immune function,and pathological examination were recorded.Results Compared with the control group,(1)food intake was decreased(P＜0.01)and body weight was increased(P＜0.05 or P＜0.01)after 90 days of continuous administration,with similar changes in the medium and high dose groups in both rats and dogs.In addition,MONO/MONO％,RBC,MCH,MCHC,HCT,HGB,PLT,TP,ALB,GLB,and Fbg were all decreased(P＜0.05 or P＜0.01)and coagulation indexes(e.g.,APTT)were increased(P＜0.05 or P＜0.01).Organ weights and the organ-to-body/brain weight ratios of the liver and spleen were increased(P＜0.05 or P＜0.01),and histopathology indicated numerous foam-like macrophages in the hepatic sinuses,red spleen pulp,and lymph node medulla.DBIL and TBIL also increased in rats in the high dose group(P＜0.05 or P＜0.01),while the dogs experienced skin swelling or scabs,abdominal swelling,vomiting,decreased activity,high albuminuria,and ascites,and the renal glomerular cells showed vacuoles.(2)After 28 days of recovery,rats and dogs in the medium and high dose groups showed a few foam-like macrophages in the hepatic sinuses,red spleen pulp,and lymph node medulla,as well as decreased of food intake in dogs.The MCHC,PLT,and TP decreased in dogs in the high dose group(P＜0.05),and the liver and spleen weights and organ coefficients in rats increased(P＜0.05 or P＜0.01),while the MONO％decreased in male rats in the medium dose group(P＜0.05).Conclusions Administration of mPEG-PLA 210 and 700 mg/kg for 90 days caused blood mononuclear cells to enter and aggregate in the liver,spleen,lymph nodes,and other tissues in SD rats and Beagle dogs,leading to secondary tissue structural damage.Protein and fibrinogen synthesis and bilirubin metabolism in the liver decreased,leading to abnormal coagulation function,and decreased intravascular colloid osmotic pressure resulted in edema and bleeding.The result suggest that the liver,spleen,kidney,and lymph nodes are target organs for mPEG-PLA toxicity,with dose-dependent and reversible effects and species differences,but no significant sex differences.Clinical monitoring of related organ functions is needed to avoid secondary damage.

ObjectiveTo provide a reference for the diagnosis of amyloidosis in experimental animals through the pathological diagnosis of systemic amyloidosis in a case of a New Zealand white rabbit. MethodsIn a 6-month repeated ocular toxicity study, an abnormal finding was noted during the routine gross anatomical examination of one New Zealand white rabbit. Its organs were prepared as paraffin sections and stained with hematoxylin-eosin (HE) staining and Congo red staining. The histopathological features were observed under optical and polarized light microscopy. ResultsGross anatomical examination of the animal revealed an enlarged spleen and changes in the color and texture of the lung. HE staining showed that the splenic tissue structure was destroyed, the white pulp of the spleen was surrounded by dense amyloid deposition in the form of nodular rings, along with pressure atrophy of the white pulp. Amyloid deposits were also observed in the submandibular lymph nodes, mesenteric lymph nodes, ileum, sacculus rotundus, vermiform appendix, jejunum, cecum, and rectum. Congo red staining showed that the amyloid deposition in the affected organs appeared salmon-pink, and exhibited characteristic apple green birefringence under polarized light microscopy.Conclusion The histo-pathological features of the New Zealand white rabbit are consistent with the diagnostic characteristics of systemic amyloidosis.

Background and Objectives: Quantitative flow ratio (QFR) is an angiography-based technique for functional assessment of coronary artery stenosis. This study investigated the response of QFR to different degree of stenosis severity and its ability to predict the positron emission tomography (PET)-defined myocardial ischemia. Methods: From 109 patients with 185 vessels who underwent both 13 N-ammonia PET and invasive physiological measurement, we compared QFR, fractional flow reserve (FFR) and instantaneous wave-free ratio (iFR) for the responses to the different degree of anatomical (percent diameter stenosis [%DS]) and hemodynamic (relative flow reserve [RFR], coronary flow reserve, hyperemic stenosis resistance, and stress myocardial flow) stenosis severity and diagnostic performance against PET-derived parameters. Results: QFR, FFR, and iFR showed similar responses to both anatomic and hemodynamic stenosis severity. Regarding RFR, the diagnostic accuracy of QFR was lower than FFR (76.2% vs. 83.2%, p=0.021) and iFR (76.2% vs. 84.3%, p=0.031). For coronary flow capacity (CFC), QFR showed a lower accuracy than iFR (74.1% vs. 82%, p=0.031) and lower discriminant function than FFR (area under curve: 0.74 vs. 0.79, p=0.044). Discordance between QFR and FFR or iFR was shown in 14.6% of cases and was driven by the difference in %DS and heterogeneous distribution of PET-derived RFR and stress myocardial blood flow. Conclusions QFR demonstrated a similar response to different anatomic and hemodynamic stenosis severity as FFR or iFR. However, its diagnostic performance was inferior to FFR and iFR when PET-derived RFR and CFC were used as a reference.

Background and Objectives: Quantitative flow ratio (QFR) is an angiography-based technique for functional assessment of coronary artery stenosis. This study investigated the response of QFR to different degree of stenosis severity and its ability to predict the positron emission tomography (PET)-defined myocardial ischemia. Methods: From 109 patients with 185 vessels who underwent both 13 N-ammonia PET and invasive physiological measurement, we compared QFR, fractional flow reserve (FFR) and instantaneous wave-free ratio (iFR) for the responses to the different degree of anatomical (percent diameter stenosis [%DS]) and hemodynamic (relative flow reserve [RFR], coronary flow reserve, hyperemic stenosis resistance, and stress myocardial flow) stenosis severity and diagnostic performance against PET-derived parameters. Results: QFR, FFR, and iFR showed similar responses to both anatomic and hemodynamic stenosis severity. Regarding RFR, the diagnostic accuracy of QFR was lower than FFR (76.2% vs. 83.2%, p=0.021) and iFR (76.2% vs. 84.3%, p=0.031). For coronary flow capacity (CFC), QFR showed a lower accuracy than iFR (74.1% vs. 82%, p=0.031) and lower discriminant function than FFR (area under curve: 0.74 vs. 0.79, p=0.044). Discordance between QFR and FFR or iFR was shown in 14.6% of cases and was driven by the difference in %DS and heterogeneous distribution of PET-derived RFR and stress myocardial blood flow. Conclusions QFR demonstrated a similar response to different anatomic and hemodynamic stenosis severity as FFR or iFR. However, its diagnostic performance was inferior to FFR and iFR when PET-derived RFR and CFC were used as a reference.