Description of a Nonhuman Primate Model of Retinal Ischemia/Reperfusion Injury.

Purpose: To establish an inducible model of retinal ischemia/reperfusion injury (RI/RI) in nonhuman primates (NHPs) to improve our understanding of the disease conditions and evaluate treatment interventions in humans. Methods: We cannulated the right eye of rhesus macaques with a needle attached to a normal saline solution reservoir at up to 1.9 m above the eye level that resulted in high intraocular pressure of over 100 mm Hg for 90 minutes. Retinal morphology and function were monitored before and after RI/RI over two months by fundus photography, optical coherence tomography, electroretinography, and visual evoked potential. Terminal experiments involved immunostaining for retinal ganglion cell marker Brn3a, glial fibrillary acidic protein, and quantitative polymerase chain reaction to assess retinal inflammatory biomarkers. Results: We observed significant and progressive declines in retinal and retinal nerve fiber layer thickness in the affected eye after RI/RI. We noted significant reductions in amplitudes of electroretinography a-wave, b-wave, and visual evoked potential N2-P2, with minimal recovery at 63 days after injury. Terminal experiments conducted two months after injury revealed ∼73% loss of retinal ganglion cells and a fivefold increase in glial fibrillary acid protein immunofluorescence intensity compared to the uninjured eyes. We observed marked increases in tumor necrosis factor-alpha, interferon-gamma, interleukin-1beta, and inducible nitric oxide synthase in the injured retinas. Conclusions: The results demonstrated that the pathophysiology observed in the NHP model of RI/RI is comparable to that of human diseases and suggest that the NHP model may serve as a valuable tool for translating interventions into viable treatment approaches. Translational Relevance: The model serves as a useful platform to study potential interventions and treatments for RI/RI or blinding retinal diseases.

Corrigendum: Integrated network pharmacology and metabolomics to reveal the mechanism of QiShenYiQi Dripping Pills against cardiac structural and functional abnormalities.

[This corrects the article DOI: 10.3389/fphar.2022.1017433.].

Integrated network pharmacology and metabolomics to reveal the mechanism of QiShenYiQi Dripping Pills (T101) against cardiac structural and functional abnormalities.

Background: Heart failure (HF), the final stage of cardiovascular diseases, is a clinical syndrome of cardiac structural or functional abnormalities. QiShenYiQi Dripping Pills (T101), short for QSYQ (T101), showed effectiveness and safety in the treatment of HF according to modern pharmacological research and clinical studies, but the mechanism remains unclear. This study aims to clarify the mechanism of QSYQ (T101) in treating heart failure through the analysis to critical biomarkers, targets and pathways. Materials and Methods: In this study, the efficacies of QSYQ (T101) in non-human primates and rodents were evaluated, and the mechanism was demonstrated by integrating network pharmacology and metabolomics analysis. Furthermore, the targets from network pharmacology and the metabolites from targeted metabolomics were jointly analyzed to screen the critical pathways. Results: In rhesus monkeys with spontaneous chronic heart failure, nasogastric administration of QSYQ (T101) for 12 weeks caused profound improvement of systolic and diastolic function as evidenced by echocardiography detection. Consistently, QSYQ (T101) administration especially with higher dose lowered the blood pressure and improved the ventricular remodeling, collagen deposition and fibrosis markedly in Spontaneous Hypertension Rats (SHR) model. Computational prediction showed that QSYQ (T101) exhibited anti-HF effects possibly through HIF-1 signaling pathway, FoxO signaling pathway, TNF signaling pathway, PI3K-Akt signaling pathway and other enriched paths. Metabolomics analysis obtained 23 significantly altered metabolites, revealing that QSYQ (T101) significantly regulated the abnormal levels of fatty acids, carnitines, organic acids pyridines, nucleosides, which were mostly involved in myocardial energy metabolism related pathways. Conclusion: Based on serum and myocardium metabolomics and network pharmacology, the present study revealed that the actions of QSYQ (T101) in treating HF depend on multi-components, multi-targets and multi-pathways.

A new non-human primate model of desiccating stress-induced dry eye disease.

Dry eye disease (DED), a multifactorial ocular surface disease, is estimated to affect up to 34% of individuals over 50 years old. Although numerous animal models, including rodents and rabbits, have been developed to mimic the pathophysiologic mechanisms involved in dry eye, there is a lack of non-human primate (NHP) models, critical for translational drug studies. Here, we developed a novel desiccating stress-induced dry eye disease model using Rhesus macaque monkeys. The monkeys were housed in a controlled environment room for 21 to 36 days under humidity, temperature, and airflow regulation. Following desiccating stress, NHPs demonstrated clinical symptoms similar to those of humans, as shown by increased corneal fluorescein staining (CFS) and decreased tear-film breakup time (TFBUT). Moreover, corticosteroid treatment significantly reduced CFS scoring, restored TFBUT, and prevented upregulation of tear proinflammatory cytokines as observed in dry eye patients following steroid treatment. The close resemblance of clinical symptoms and treatment responses to those of human DED patients provides great translational value to the NHP model, which could serve as a clinically relevant animal model to study the efficacy of new potential treatments for DED.

Development of Primary Open Angle Glaucoma-Like Features in a Rhesus Macaque Colony From Southern China.

Purpose: To describe the ocular phenotype of spontaneous glaucoma in a non-human primate colony. Methods: In total, 722 Rhesus macaque monkeys aged 10 to 25 years underwent optical coherence tomography (OCT), fundus photography (FP), and intraocular pressure (IOP) measurements. Monkeys with baseline cup-to-disc ratio (CDR) <0.5 were used to establish baseline ocular features. A subset was followed longitudinally for three years and compared to glaucoma suspects on the basis of OCT/FP criteria. Results: The average IOP under ketamine sedation and average CDR for the entire colony was 13.0 ± 4.3 mm Hg and 0.38 ± 0.07, respectively. The mean baseline conscious IOP of glaucoma suspects (N = 18) versus controls (N = 108) was 16.2 ± 3.5 mm Hg and 13.9 ± 2.3 mm Hg, respectively (P = 0.001). All glaucoma suspects had unremarkable slit lamp examinations and open angles based on anterior segment OCT. Baseline global circumpapillary retinal nerve fiber layer (RNFL) thickness was 91.5 ± 11.0 µM versus 102.7 ± 8.5 µM in suspects and controls, respectively (P < 0.0001). All sectors on the baseline circumpapillary OCT showed a significant reduction in RNFL thickness versus controls (P ≤ 0.0022) except for the temporal sector (P ≥ 0.07). In three-year longitudinal analysis, neither CDR nor OCT parameters changed in controls (N = 40; P ≥ 0.16), whereas significant increase in CDR (P = 0.018) and nominally significant decreases in two OCT sectors (nasal, P = 0.023 and nasal inferior, P = 0.046) were noted in suspects. Conclusions: Members of a nonhuman primate colony exhibit important ophthalmic features of human primary open-angle glaucoma. Translational Relevance: Identification of a spontaneous model of glaucoma in nonhuman primates represents an unprecedented opportunity to elucidate the natural history, pathogenesis and effective therapeutic strategies for the disease.

Novel GLP-1 analog supaglutide improves glucose homeostasis in diabetic monkeys.

Glucagon-like peptide 1 (GLP-1) is an insulinotropic hormone and plays an important role in regulating glucose homeostasis. GLP-1 has a short half-life (t1/2 < 2 min) due to degrading enzyme dipeptidyl peptidase-IV and rapid kidney clearance, which limits its clinical application as a therapeutic reagent. We demonstrated recently that supaglutide, a novel GLP-1 mimetic generated by recombinant fusion protein techniques, exerted hypoglycemic and ß-cell trophic effects in type 2 diabetes db/db mice. In the present study, we examined supaglutide's therapeutic efficacy and pharmacokinetics in diabetic rhesus monkeys. We found that a single subcutaneous injection of supaglutide of tested doses transiently and significantly reduced blood glucose levels in a dose-dependent fashion in the diabetic monkeys. During a 4-week intervention period, treatment of supaglutide of weekly dosing dose-dependently decreased fasting and random blood glucose levels. This was associated with significantly declined plasma fructosamine levels. The repeated administration of supaglutide remarkably also decreased body weight in a dose-dependent fashion accompanied by decreased food intake. Intravenous glucose tolerance test results showed that supaglutide improved glucose tolerance. The intervention also showed enhanced glucose-stimulated insulin secretion and improved lipid profile in diabetic rhesus monkeys. These results reveal that supaglutide exerts beneficial effects in regulating blood glucose and lipid homeostasis in diabetic rhesus monkeys.

Evaluating the correlation of the impairment between skeletal muscle and heart using MRI in a spontaneous type 2 diabetes mellitus rhesus monkey model.

AIMS: To investigate the correlation of impairment in skeletal muscle and heart in spontaneous type 2 diabetes mellitus (T2DM) rhesus monkeys using magnetic resonance image (MRI). METHODS: Fifteen T2DM monkeys and fourteen healthy control (HC) monkeys were included. The microcirculation of skeletal muscle [skeletal muscle blood flow (SMBF), skeletal muscle oxygen extraction fraction (SMOEF)] and the function and strain of heart were evaluated by MRI. Three regions of interests were chosen on the soleus muscle (SOL), gastrocnemius muscle (GAS) and tibialis anterior muscle (TA) for image analysis. RESULTS: Eight T2DM monkeys and eight HC monkeys were obtained the full data. The SMBF reserves and SMOEF reserves were found significantly decreased in T2DM during inflation in SOL, GAS and TA muscles (all p < 0.05), and the SMBF reserves decreased during hyperemia in GAS and TA muscles (all p < 0.05). In these monkeys, the global peak longitudinal strain (longitudinal PS), peak systolic longitudinal strain rate (longitudinal PSSR) and peak diastolic longitudinal strain rate (longitudinal PDSR) were seen significantly different in T2DM compared to HC monkeys (all p < 0.05). The longitudinal PSSR was found negatively correlated with SMBF reserves in SOL, GAS and TA during inflation in all monkeys. CONCLUSIONS: The impaired microcirculation of skeletal muscle and the myocardial deformation were found in T2DM monkeys with normal ejection fraction. And a negative correlation was existed in the longitudinal PSSR and the SMBF reserves.

2D/3D CMR tissue tracking versus CMR tagging in the assessment of spontaneous T2DM rhesus monkeys with isolated diastolic dysfunction.

BACKGROUND: Spontaneous T2DM in rhesus monkeys manifests as isolated diastolic dysfunction in the early stage of diabetic cardiomyopathy, similar to humans. Myocardial deformation measurements have emerged as a superior way to measure left ventricular (LV) function in the early stage of cardiac dysfunction, making it possible to further evaluate early-stage LV dysfunction in spontaneous T2DM rhesus monkeys. METHODS: Spontaneous T2DM rhesus monkeys with isolated diastolic dysfunction (T2DM-DD, n = 10) and corresponding nondiabetic healthy animals (ND, n = 9) were prospectively scanned for a CMR study. Circumferential and longitudinal peak systolic strain (Ecc, Ell), time to peak strain (tEcc, tEll) and peak diastolic strain rate (CSR, LSR) obtained from 2D/3D CMR-TT were compared with those obtained from CMR tagging separately. In addition, all CMR imaging protocols were performed twice in 9 ND animals to assess test-retest reproducibility. RESULTS: Compared with the ND group, the T2DM-DD monkeys demonstrated significantly impaired LV Ecc (- 10.63 ± 3.23 vs - 14.18 ± 3.19, p < 0.05), CSR (65.50 ± 14.48 vs 65.50 ± 14.48, p < 0.01), Ell (- 9.11 ± 2.59 vs - 14.17 ± 1.68, p < 0.05), and LSR (59.43 ± 19.17 vs 108.46 ± 22.33, p < 0.01) with the tagging. Only Ecc (- 13.10 ± 2.47 vs - 19.03 ± 3.69, p < 0.01) and CSR (148.90 ± 31.27 vs 202.00 ± 51.88, p < 0.01) were significantly reduced with 2D CMR-TT, and only Ecc (- 13.77 ± 1.98 vs - 17.26 ± 3.78, p < 0.05) was significantly reduced with 3D CMR-TT. Moreover, 2D/3D CMR-TT-derived Ecc and CSR correlated with the corresponding tagging values collectively, with a statistically significant ICC value (p < 0.05). Test-retest repeatability analysis showed that most tagging-derived biomarkers had acceptable repeatability (p < 0.01). In addition, 2D CMR-TT-derived indicators were poorer than those derived from the tagging method but better than those obtained using the 3D method, with larger ICCs except for tEcc (p < 0.05). CONCLUSIONS: LV systolic and diastolic deformations were impaired in spontaneous T2DM rhesus monkeys previously diagnosed with isolated diastolic dysfunction by echocardiography. The 2D CMR-TT-derived Ecc and CSR were effective in the evaluation of the myocardial systolic and diastolic functions of early-diabetic cardiomyopathy, with relatively higher test-retest reproducibility and acceptable correlation with the tagging method compared with the 3D CMR-TT method.

Anti-hypercholesterolemic Effects and a Good Safety Profile of SCM-198 in Animals: From ApoE Knockout Mice to Rhesus Monkeys.

Although several lipid-lowering agents have been introduced for the treatment of atherosclerosis (AS), currently marketed medications have not solved the problem completely. This study aims to investigate the effects of leonurine (SCM-198) on dyslipidemia in mammals with ApoE knockout (ApoE-/-) mice, New Zealand white rabbits and senile Rhesus monkeys fed with high fat diet were dosed daily with leonurine or atorvastatin. The serum total cholesterol (TC), triglyceride (TG), low density lipoprotein (LDL), and high-density lipoprotein (HDL) were determined. Moreover, in Rhesus monkeys, bodyweight, arterial ultrasound of right common carotid artery, Apolipoprotein A1 (ApoA1) and ApoB levels, hematologic and toxicological examinations were detected. Serum TC and TG in both mice and rabbits were significantly reduced by SCM-198 and atorvastatin. In the 10 mg/kg SCM-198 group of monkeys, maximum TC reduction of 24.05% was achieved at day 150, while 13.16% LDL reduction achieved at day 60, without arterial morphologic changes or adverse events. Atorvastatin (1.2 mg/kg) showed similar effects as SCM-198 in improving lipid profiles in monkeys, yet its long-term use could induce tolerance. Furthermore, leonurine suppressed genes expression of fatty acid synthesis, such as fatty acid synthase (FASN), stearoyl-CoA desaturase (SCD-1), sterol regulatory element-binding protein (SREBF) in liver in high fat diet feeding ApoE-/- mice. SCM-198, with a reliable safety profile, is of high value in improving lipid profiles in mammals, providing an alternative to a substantial population who are statin-intolerant.

The quantification of blood-brain barrier disruption using dynamic contrast-enhanced magnetic resonance imaging in aging rhesus monkeys with spontaneous type 2 diabetes mellitus.

Microvascular lesions of the body are one of the most serious complications that can affect patients with type 2 diabetes mellitus. The blood-brain barrier (BBB) is a highly selective permeable barrier around the microvessels of the brain. This study investigated BBB disruption in diabetic rhesus monkeys using dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). Multi-slice DCE-MRI was used to quantify BBB permeability. Five diabetic monkeys and six control monkeys underwent magnetic resonance brain imaging in 3 Tesla MRI system. Regions of the frontal cortex, the temporal cortex, the basal ganglia, the thalamus, and the hippocampus in the two groups were selected as regions of interest to calculate the value of the transport coefficient Ktrans using the extended Tofts model. Permeability in the diabetic monkeys was significantly increased as compared with permeability in the normal control monkeys. Histopathologically, zonula occludens protein-1 decreased, immunoglobulin G leaked out of the blood, and nuclear factor E2-related factor translocated from the cytoplasm to the nuclei. It is likely that diabetes contributed to the increased BBB permeability.

Diastolic dysfunction in spontaneous type 2 diabetes rhesus monkeys: a study using echocardiography and magnetic resonance imaging.

BACKGROUND: Diastolic heart failure is a common and deadly complication of diabetes mellitus, with the development of diabetic cardiomyopathy as one of the key determinants of the disease's complex pathology. The cause of the association is unknown and has no approved therapy strategies as of yet. However significant advances in this area may come from studies on suitable animal models. METHODS: A total of 25 male rhesus monkeys (12-16 years, 9-13 kg) were enrolled. Fifteen of them were diagnosed as spontaneous type 2 diabetes mellitus (T2DM, FPG ≥ 104 mg/dl, HbA1c: 4.7-5.5 %, diabetes duration: 1-4 years). The other 10 monkeys were non-diabetic (ND, FPG < 90 mg/dl). Echocardiography and cardiac magnetic resonance were used for evaluating the cardiac structure and function. One T2DM monkey with impaired diastolic function and another ND monkey were both sacrificed to gain the necessary pathology and protein expression studies displayed here. RESULTS: Six out of 15 T2DM rhesus monkeys were diagnosed with diastolic dysfunction (DD) by echocardiography. Additionally, no abnormalities were found in the group which we determined as the ND monkeys. The six DD monkeys all showed low e' velocity and decreased e'/a' ratio, among which three of them showing decreased E/A ratio and the other 3 having elevated E/A ratio, this appears to be similar to the impaired relaxation pattern and pseudonormal pattern found in human patients respectively. The EF and FS of monkeys with pseudonormal pattern decreased significantly compared with ND subjects. A CMR study showed that LVID at end systole of 5 DD monkeys is significantly longer than that of 3 ND monkeys. Of great interest, myocardium lesions and mitochondria impairments and increased expression of AGEs and caspase-3 were found in a sacrificed DD subject. CONCLUSION: The changes in the imaging and physiological markers of spontaneous T2DM rhesus monkeys are similar to those key markers found in human type 2 diabetes and diastolic dysfunction. This monkey model could help the medical community and us to understand the pathology of this debilitating disease and serve as a beginning to explore important measures to prevent and treat diabetic cardiomyopathy.

Comparison of the clinical manifestations of type 2 diabetes mellitus between rhesus monkey (Macaca mulatta lasiotis) and human being.

OBJECTIVES: Type 2 diabetes mellitus (T2DM) is the most common form of diabetes. To determine the similarities of development of T2DM between rhesus monkey [Macaca mulatta lasiotis (M. m. lasiotis)] and human being, the clinical parameters were determined during a period of 2 years in 60 adult male rhesus monkeys (M. m. lasiotis). METHODS: Sixty male monkeys whose fasting plasma glucose (FPG) level less than or equal to 5 mmol/L (90 mg/dL) were enrolled in this study. Of these, 50 monkeys aged 7 to 20 years were fed with high-fat diet and 10 aged 4 to 10 years fed with standard diet as normal monkeys. Body weight, body mass index, FPG, fasting plasma insulin, and hemoglobin A1c levels were measured and calculated. The responses of insulin and glucose levels to intravenous glucose tolerance test were analyzed. RESULTS: Of 50 monkeys fed with high-fat diet, 8 developed overt T2DM, 26 experienced impaired glucose tolerance and impaired fasting glucose, and FPG of 16 monkeys was normal. All monkeys with impaired glucose tolerance experienced obesity, compensatory increase of fasting plasma insulin, significant decline of postprandial glucose clearance rate (KGluc5-20), and decreased insulin secretion. CONCLUSIONS: In conclusion, rhesus monkey (M. m. lasiotis) has many similarities with human beings in terms of clinical manifestations and risk factors at different diabetes stages.