Small-Molecule-Directed Endogenous Regeneration of Visual Function in a Mammalian Retinal Degeneration Model.

Degenerative retinal diseases associated with photoreceptor loss are a leading cause of visual impairment worldwide, with limited treatment options. Phenotypic profiling coupled with medicinal chemistry were used to develop a small molecule with proliferative effects on retinal stem/progenitor cells, as assessed in vitro in a neurosphere assay and in vivo by measuring Msx1-positive ciliary body cell proliferation. The compound was identified as having kinase inhibitory activity and was subjected to cellular pathway analysis in non-retinal human primary cell systems. When tested in a disease-relevant murine model of adult retinal degeneration (MNU-induced retinal degeneration), we observed that four repeat intravitreal injections of the compound improved the thickness of the outer nuclear layer along with the regeneration of the visual function, as measured with ERG, visual acuity, and contrast sensitivity tests. This serves as a proof of concept for the use of a small molecule to promote endogenous regeneration in the eye.

Activation of adult mammalian retinal stem cells in vivo via antagonism of BMP and sFRP2.

BACKGROUND: The adult mammalian retina does not have the capacity to regenerate cells lost due to damage or disease. Therefore, retinal injuries and blinding diseases result in irreversible vision loss. However, retinal stem cells (RSCs), which participate in retinogenesis during development, persist in a quiescent state in the ciliary epithelium (CE) of the adult mammalian eye. Moreover, RSCs retain the ability to generate all retinal cell types when cultured in vitro, including photoreceptors. Therefore, it may be possible to activate endogenous RSCs to induce retinal neurogenesis in vivo and restore vision in the adult mammalian eye. METHODS: To investigate if endogenous RSCs can be activated, we performed combinatorial intravitreal injections of antagonists to BMP and sFRP2 proteins (two proposed mediators of RSC quiescence in vivo), with or without growth factors FGF and Insulin. We also investigated the effects of chemically-induced N-methyl-N-Nitrosourea (MNU) retinal degeneration on RSC activation, both alone and in combination withthe injected factors. Further, we employed inducible Msx1-CreERT2 genetic lineage labeling of the CE followed by stimulation paradigms to determine if activated endogenous RSCs could migrate into the retina and differentiate into retinal neurons. RESULTS: We found that in vivo antagonism of BMP and sFRP2 proteins induced CE cells in the RSC niche to proliferate and expanded the RSC population. BMP and sFRP2 antagonism also enhanced CE cell proliferation in response to exogenous growth factor stimulation and MNU-induced retinal degeneration. Furthermore, Msx1-CreERT2 genetic lineage tracing revealed that CE cells migrated into the retina following stimulation and/or injury, where they expressed markers of mature photoreceptors and retinal ganglion cells. CONCLUSIONS: Together, these results indicate that endogenous adult mammalian RSCs may have latent regenerative potential that can be activated by modulating the RSC niche and hold promise as a means for endogenous retinal cell therapy to repair the retina and improve vision.

Glucocorticoid agonists enhance retinal stem cell self-renewal and proliferation.

BACKGROUND: Adult mammalian retinal stem cells (RSCs) readily proliferate, self-renew, and generate progeny that differentiate into all retinal cell types in vitro. RSC-derived progeny can be induced to differentiate into photoreceptors, making them a potential source for retinal cell transplant therapies. Despite their proliferative propensity in vitro, RSCs in the adult mammalian eye do not proliferate and do not have a regenerative response to injury. Thus, identifying and modulating the mechanisms that regulate RSC proliferation may enhance the capacity to produce RSC-derived progeny in vitro and enable RSC activation in vivo. METHODS: Here, we used medium-throughput screening to identify small molecules that can expand the number of RSCs and their progeny in culture. In vitro differentiation assays were used to assess the effects of synthetic glucocorticoid agonist dexamethasone on RSC-derived progenitor cell fate. Intravitreal injections of dexamethasone into adult mouse eyes were used to investigate the effects on endogenous RSCs. RESULTS: We discovered that high-affinity synthetic glucocorticoid agonists increase RSC self-renewal and increase retinal progenitor proliferation up to 6-fold without influencing their differentiation in vitro. Intravitreal injection of synthetic glucocorticoid agonist dexamethasone induced in vivo proliferation in the ciliary epithelium-the niche in which adult RSCs reside. CONCLUSIONS: Together, our results identify glucocorticoids as novel regulators of retinal stem and progenitor cell proliferation in culture and provide evidence that GCs may activate endogenous RSCs.

Induction of rod versus cone photoreceptor-specific progenitors from retinal precursor cells.

During development, multipotent progenitors undergo temporally-restricted differentiation into post-mitotic retinal cells; however, the mechanisms of progenitor division that occurs during retinogenesis remain controversial. Using clonal analyses (lineage tracing and single cell cultures), we identify rod versus cone lineage-specific progenitors derived from both adult retinal stem cells and embryonic neural retinal precursors. Taurine and retinoic acid are shown to act in an instructive and lineage-restricted manner early in the progenitor lineage hierarchy to produce rod-restricted progenitors from stem cell progeny. We also identify an instructive, but lineage-independent, mechanism for the specification of cone-restricted progenitors through the suppression of multiple differentiation signaling pathways. These data indicate that exogenous signals play critical roles in directing lineage decisions and resulting in fate-restricted rod or cone photoreceptor progenitors in culture. Additional factors may be involved in governing photoreceptor fates in vivo.

Aerobic Endurance Training Does Not Protect Bone Against Poorly Controlled Type 1 Diabetes in Young Adult Rats.

Streptozotocin (STZ)-induced type 1 diabetes mellitus (T1DM) decreases trabecular bone volume and bone strength in rodents. The current study investigated the potential protective effects of aerobic endurance training (AET) on bone in STZ-induced T1DM young adult rats. Sixty-four 8-week-old male Sprague-Dawley rats were randomly divided into 4 groups of 16: control non-T1DM sedentary (CS) and exercised (CX), T1DM sedentary (DS) and exercised (DX). Blood glucose was maintained at 9-15 mmol/L using subcutaneously implanted insulin pellets (Linplant, Linshin Canada, Inc.). AET was performed at ~75-85% VO2max for 1 h/day, 5 day/week for 10 weeks. Areal and volumetric bone mineral density (aBMD and vBMD; excised femur) were measured using dual-energy X-ray absorptiometry (DXA; QDR 4500A) and micro computed tomography (µCT; Aloka). Bone strength was tested using a 3-point bending test (Instron 5544 Load Frame). Two-way ANOVA was used to test for T1DM and exercise differences followed by Tukey's HSD tests for interaction effects; significance was set at P < 0.05. T1DM had lower body weight (18.0%), aBMD (8.6%), cortical vBMD (1.6%), trabecular vBMD (2.1%), maximum load at break (22.2%), and increased elastic modulus (11.3%) vs. control (P < 0.001). Exercise in T1DM further decreased body weight (4.7%) vs. sedentary (P = 0.043) and maximum extension during the bending test that demonstrated DX was increased (7.3%) vs. CX (P = 0.033). There were no other beneficial effects of exercise on bone. These results suggest that 10 weeks of AET in rats do not have protective effects on bone in the short term and that T1DM rats have compromised bone health.

The glucoregulatory response to high-intensity aerobic exercise following training in rats with insulin-treated type 1 diabetes mellitus.

An acute bout of exercise elicits a rapid, potentially deleterious, reduction in blood glucose in patients with type 1 diabetes mellitus (T1DM). In the current study, we examined whether a 10-week aerobic training program could alleviate the rapid exercise-associated reduction in blood glucose through changes in the glucoregulatory hormonal response or increased hepatic glycogen storage in an insulin-treated rat model of T1DM. Thirty-two male Sprague-Dawley rats were divided evenly into 4 groups: non-T1DM sedentary (C) (n = 8), non-T1DM exercised (CX) (n = 8), T1DM sedentary (D) (n = 8), and T1DM exercised (DX) (n = 8). Exercise training consisted of treadmill running for 5 days/week (1 h, 27 m/min, 6% grade) for 10 weeks. T1DM was induced by multiple streptozotocin injections (20 mg/kg) followed by implantation of subcutaneous insulin pellets. At week 1, an acute exercise bout led to a significant reduction in blood glucose in DX (p < 0.05), whereas CX exhibited an increase in blood glucose (p < 0.05). During acute exercise, serum epinephrine was increased in both DX and CX (p < 0.05), whereas serum glucagon was increased during recovery only in CX (p < 0.01). Following aerobic training in DX, the exercise-mediated reduction in blood glucose remained; however, serum glucagon increased to the same extent as in CX (p < 0.05). DX exhibited significantly less hepatic glycogen (p < 0.001) despite elevations in glycogenic proteins in the liver (p < 0.05). Elevated serum epinephrine and decreased hepatic adrenergic receptor expression were also evident in DX (p < 0.05). In summary, despite aerobic training in DX, abrupt blood glucose reductions and hepatic glycogen deficiencies were evident. These data suggest that sympathetic overactivity may contribute to deficiencies in hepatic glycogen storage.

High Intensity Aerobic Exercise Training Improves Deficits of Cardiovascular Autonomic Function in a Rat Model of Type 1 Diabetes Mellitus with Moderate Hyperglycemia.

Indices of cardiovascular autonomic neuropathy (CAN) in experimental models of Type 1 diabetes mellitus (T1DM) are often contrary to clinical data. Here, we investigated whether a relatable insulin-treated model of T1DM would induce deficits in cardiovascular (CV) autonomic function more reflective of clinical results and if exercise training could prevent those deficits. Sixty-four rats were divided into four groups: sedentary control (C), sedentary T1DM (D), control exercise (CX), or T1DM exercise (DX). Diabetes was induced via multiple low-dose injections of streptozotocin and blood glucose was maintained at moderate hyperglycemia (9-17 mM) through insulin supplementation. Exercise training consisted of daily treadmill running for 10 weeks. Compared to C, D had blunted baroreflex sensitivity, increased vascular sympathetic tone, increased serum neuropeptide Y (NPY), and decreased intrinsic heart rate. In contrast, DX differed from D in all measures of CAN (except NPY), including heart rate variability. These findings demonstrate that this T1DM model elicits deficits and exercise-mediated improvements to CV autonomic function which are reflective of clinical T1DM.

The relationship between blood pressure and sciatic nerve blood flow velocity in rats with insulin-treated experimental diabetes.

Peripheral nerve blood flow (NBF) does not autoregulate but, instead, responds passively to changes in mean arterial pressure (MAP). How this relationship is impacted by insulin-treated experimental diabetes (ITED) is unknown. We tested the hypothesis that ITED will reduce NBF across a range of MAP in Sprague Dawley rats. Following 10 weeks of control or ITED conditions, conscious MAP (tail-cuff) was measured, and under anaesthesia, the MAP (carotid artery catheter, pressure transducer) and NBF (Doppler ultrasound, 40 MHz) responses to sodium nitroprusside (60 µg/kg) and phenylephrine (12 µg/kg) infusion were recorded (regression equations for MAP vs NBF were created for each rodent). Thereafter, motor nerve conduction velocity (MNCV) and nerve vascularization (haematoxylin and eosin stain) were determined. Conscious MAP was higher and MNCV was lower in the ITED group (p < 0.01). In response to drug infusions, the ΔMAP and ΔNBF were similar between groups (p ≥ 0.18). Estimated conscious NBF (based on substituting conscious MAP values into each individual regression equation) was greater in the ITED group (p < 0.01). Sciatic nerve vascularization was similar between groups (p ≥ 0.50). In contrast to the hypothesis, NBF was not reduced across a range of MAP. In spite of increased estimated conscious NBF values, MNCV was reduced in rats with ITED.

Exercise training enhances insulin-stimulated nerve arterial vasodilation in rats with insulin-treated experimental diabetes.

Insulin stimulates nerve arterial vasodilation through a nitric oxide (NO) synthase (NOS) mechanism. Experimental diabetes reduces vasa nervorum NO reactivity. Studies investigating hyperglycemia and nerve arterial vasodilation typically omit insulin treatment and use sedentary rats resulting in severe hyperglycemia. We tested the hypotheses that 1) insulin-treated experimental diabetes and inactivity (DS rats) will attenuate insulin-mediated nerve arterial vasodilation, and 2) deficits in vasodilation in DS rats will be overcome by concurrent exercise training (DX rats; 75-85% VO2 max, 1 h/day, 5 days/wk, for 10 wk). The baseline index of vascular conductance values (VCi = nerve blood flow velocity/mean arterial blood pressure) were similar (P ≥ 0.68), but peak VCi and the area under the curve (AUCi) for the VCi during a euglycemic hyperinsulinemic clamp (EHC; 10 mU·kg(-1)·min(-1)) were lower in DS rats versus control sedentary (CS) rats and DX rats (P ≤ 0.01). Motor nerve conduction velocity (MNCV) was lower in DS rats versus CS rats and DX rats (P ≤ 0.01). When compared with DS rats, DX rats expressed greater nerve endothelial NOS (eNOS) protein content (P = 0.04). In a separate analysis, we examined the impact of diabetes in exercise-trained rats alone. When compared with exercise-trained control rats (CX), DX rats had a lower AUCi during the EHC, lower MNCV values, and lower sciatic nerve eNOS protein content (P ≤ 0.03). Therefore, vasa nervorum and motor nerve function are impaired in DS rats. Such deficits in rats with diabetes can be overcome by concurrent exercise training. However, in exercise-trained rats (CX and DX groups), moderate hyperglycemia lowers vasa nervorum and nerve function.

The role of resistance and aerobic exercise training on insulin sensitivity measures in STZ-induced Type 1 diabetic rodents.

UNLABELLED: Individuals with Type 1 Diabetes Mellitus (T1DM) can develop insulin resistance. Regular exercise may improve insulin resistance partially through increased expression of skeletal muscle GLUT4 content. OBJECTIVE: To examine if different exercise training modalities can alter glucose tolerance through changes in skeletal muscle GLUT4 content in T1DM rats. METHODS: Fifty rats were divided into 5 groups; control, diabetic control, diabetic resistance exercised, and diabetic high and low intensity treadmill exercised. Diabetes was induced using multiple low dose Streptozotocin (20 mg/kg/day) injections and blood glucose concentrations were maintained moderately hyperglycemic through subcutaneous insulin pellets. Resistance trained rats climbed a ladder with incremental loads, while treadmill trained rats ran on a treadmill at 27 or 15 m/min, respectively, all for 6 weeks. RESULTS: At weeks 3 and 6, area under the curve measurements following an intravenous glucose tolerance test (AUC-IVGTT) in all diabetic groups were higher than control rats (p<0.05). At 6 weeks, all exercise groups had significantly lower AUC-IVGTT values than diabetic control animals (p<0.05). Treadmill trained rats had the lowest insulin dose requirement of the T1DM rats and the greatest reduction in insulin dosage was evident in high intensity treadmill exercise. Concomitant with improvements in glucose handling improvements, tissue-specific elevations in GLUT4 content were demonstrated in both red and white portions of vastus lateralis and gastrocnemius muscles, suggesting that glucose handling capacity was altered in the skeletal muscle of exercised T1DM rats. CONCLUSIONS: These results suggest that, while all exercise modalities can improve glucose tolerance, each mode leads to differential improvements in insulin requirements and protein content alterations.

Glucose-stimulated insulin secretion causes an insulin-dependent nitric oxide-mediated vasodilation in the blood supply of the rat sciatic nerve.

This study tested the hypothesis that acute hyperglycemia reduces sciatic nerve blood flow in Sprague-Dawley rats. Anesthetized rats underwent cannulation of their right jugular vein (for anesthetic/nutrient/drug infusion) and right carotid artery (for continuous blood pressure measurement via pressure transducer). The left sciatic nerve was exposed and nerve blood velocity (NBV) was assessed from an arterial segment lying superficially along the sciatic nerve (Doppler ultrasound, 40 MHz). NBV and mean arterial pressure (MAP) values were collected, and an index of nerve vascular conductance (NVC) was established (NBV/MAP) at baseline and at 5, 10, 20, and 30 min (and 80 min for insulin) following 1) low glucose infusion, 1 g/kg (50% solution); 2) high glucose infusion, 3 g/kg; 3) high glucose infusion in the absence of a functioning pancreas; 4) euglycemic hyperinsulinemic clamp-insulin infusion (10 mU·kg⁻¹·min⁻¹; 0.4 IU/ml); 5) high glucose infusion + NG-nitro-L-arginine methyl ester (L-NAME) infusion (30 mg/kg); and 6) L-NAME alone followed 20 min later by high glucose infusion. High glucose infusion increased NVC by ~120% relative to baseline (P < 0.001), and this dilation was attenuated in rats without a functioning pancreas (i.e., without insulin secretion) (P = 0.004) and following L-NAME infusion (P = 0.011). Therefore, the vasodilation in rat sciatic nerve during glucose infusion was dependent upon the insulin response and acted through a nitric oxide synthase pathway.

Acute endurance exercise induces changes in vasorelaxation responses that are vessel-specific.

The dynamic adjustment and amplitude of the endothelium-dependent vasorelaxation of the carotid, aorta, iliac, and femoral vessels were measured in response to acute low- (LI) or high-intensity (HI) endurance exercise. Vasorelaxation to 10(-4) M ACh was evaluated in 10 control, 10 LI, and 10 HI rats. Two-millimeter sections of carotid, aorta, iliac, and femoral arteries were mounted onto a myography system. Vasorelaxation responses were modeled as a monoexponential function. The overall τ (control, 10.5 ± 6.0 s; LI, 10.4 ± 5.7 s; HI, 11.0 ± 6.9 s) and time-to-steady-state (control, 47.6 ± 24.0 s; LI, 46.2 ± 22.8 s; HI, 49.1 ± 28.3 s) was similar in LI, HI, and control (P > 0.05). The overall (average of four vessel-type) % vasorelaxation was larger in LI (73 ± 16%) and HI (73 ± 16%) than in control (66 ± 19%) (P < 0.05). The overall rate of vasorelaxation was greater in LI (1.9 ± 0.9%·s(-1)) and HI (1.9 ± 1.1%·s(-1)) compared with control (1.6 ± 0.7%·s(-1)) (P < 0.05). The vessel-specific responses (average response for the three conditions) showed that carotid displayed a slower adjustment (τ, 18.9 ± 4.4 s; time-to-steady-state, 80.4 ± 18.4 s) compared with the aorta (τ, 10.3 ± 3.8 s; time-to-steady-state, 46.3 ± 15.2 s), the iliac (τ, 6.3 ± 2.1 s; time-to-steady-state, 30.3 ± 9.0 s), and the femoral (τ, 6.0 ± 1.9 s; time-to-steady-state, 29.3 ± 8.4 s). The % vasorelaxation was larger in the carotid (82 ± 14%) than in the aorta (67 ± 16%), iliac (61 ± 13%), and femoral (71 ± 19%) (P > 0.05). The rate of vasorelaxation was carotid (1.1 ± 0.2%·s(-1)), aorta (1.5 ± 0.4%·s(-1)), iliac (2.2 ± 0.8%·s(-1)), and femoral (2.6 ± 1.0%·s(-1)). In conclusion, an acute bout of endurance exercise increased vascular responsiveness. The dynamic and percent adjustments were vessel-specific with vessel function likely determining the response.