Effect of compound treatments on mouse lens viscoelasticity.

Previous studies have shown that pharmaceutical agents such as lipoic acid have the ability to soften the lens, presenting a promising avenue for treating presbyopia. One obstacle encountered in the preclinical stage of such agents is the need for precise measurements of lens elasticity in experimental models. This study aimed to evaluate the effects of 25-hydroxycholesterol, lipoic acid, and obeticholic acid on the viscoelastic properties of mouse lenses using a custom-built elastometer system. Data were acquired on lenses from C57BL/6J female mice from two age groups: young (age: 8-10 weeks) and old (age: 32-43 weeks). OD lenses were used as the control and OS lenses were treated. Control lenses were immersed in Dulbecco's Modified Eagle Medium (DMEM) and treatment lenses were immersed in a compound solution containing 25-hydroxycholesterol (5 young and 5 old), lipoic acid at 2.35 mM (5 young and 5 old), lipoic acid at 0.66 mM (5 old), or obeticholic acid (5 old) at 37ºC for 18 hours. After treatment, the mouse lenses were placed in a DMEM-filled chamber within a custom-built elastometer system that recorded the load and lens shape as the lens was compressed by 600 µm at a speed of 50 µm/s. The load was continuously recorded during compression and during stress-relaxation. The compression phase was fit with a linear function to quantify lens stiffness. The stress-relaxation phase was fit with a 3-term exponential relaxation model providing relaxation time constants (t1, t2, t3), and equilibrium load. The lens stiffness, time constants and equilibrium load were compared for the control and treated groups. Results revealed an increase in stiffness with age for the control group (young: 1.16 ± 0.11 g/mm, old: 1.29 ± 0.14 g/mm) and relaxation time constants decreased with age (young: t1 = 221.9 ± 29.0 s, t2 = 24.7 ± 3.8 s, t3 = 3.12 ± 0.87 s, old: t1 = 183.0 ± 22.0 s, t2 = 20.6 ± 2.6 s and t3 = 2.24 ± 0.43 s). Among the compounds tested, only 25-hydroxycholesterol produced statistically significant changes in the lens stiffness, relaxation time constants, and equilibrium load. In conclusion, older mouse lenses are stiffer and less viscous than young mouse lenses. Notably, no significant change in lens stiffness was observed following treatment with lipoic acid, contrary to previous findings.

Sustained Alleviation of Autoimmunity by Activating α2B-adrenergic Receptors.

Catecholamines binding to α- and ß-adrenergic receptors on immune cells have recently been shown to play an important role in regulating immune responses. Although α2-adrenergic receptors are known to modulate the immune response in different ways, the therapeutic exploration of their utility has been limited by the lack of agonists selective for the three α2-adrenergic subtypes. We report in this study the identification of the agonist AGN-762, which activates α2B- and α2C-adrenergic subtypes, but not the α2A subtype. We show that AGN-762 reduced clinical disease in an experimental autoimmune encephalitis model of autoimmune disease via direct or indirect effects on T regulatory cells. The activity of AGN-762 was abrogated by depletion of T regulatory cells, which express the α2B-adrenergic receptor. Furthermore, a drug-induced shift to an anti-inflammatory phenotype was demonstrated in immune cells in the spleen of drug-treated experimental autoimmune encephalitis mice. AGN-762 does not display sedative and cardiovascular side effects associated with α2A subtype agonists. Immune modulation by selective α2-adrenergic agonists represents a novel, to our knowledge, approach for treating autoimmune disease.

Measuring the effects of postmortem time and age on mouse lens elasticity using atomic force microscopy.

The mouse lens is frequently used both in vivo and ex vivo in ophthalmic research to model conditions affecting the human lens, such as presbyopia. The mouse lens has a delicate structure which is prone to damage and biomechanical changes both before and after extraction from the whole globe. When not properly controlled for, these changes can confound the biomechanical analysis of mouse lenses. In this study, atomic force microscopy microindentation was used to assess changes in the Young's Modulus of Elasticity of the mouse lens as a function of mouse age and postmortem time. Old mouse lenses measured immediately postmortem were significantly stiffer than young mouse lenses (p = 0.028). However, after 18 h of incubation, there was no measurable difference in lens stiffness between old and young mouse lenses (p = 0.997). This demonstrates the need for careful experimental control in experiments using the mouse lens, especially regarding postmortem time.

Translational Preclinical Pharmacologic Disease Models for Ophthalmic Drug Development.

Preclinical models of human diseases are critical to our understanding of disease etiology, pathology, and progression and enable the development of effective treatments. An ideal model of human disease should capture anatomical features and pathophysiological mechanisms, mimic the progression pattern, and should be amenable to evaluating translational endpoints and treatment approaches. Preclinical animal models have been developed for a variety of human ophthalmological diseases to mirror disease mechanisms, location of the affected region in the eye and severity. These models offer clues to aid in our fundamental understanding of disease pathogenesis and enable progression of new therapies to clinical development by providing an opportunity to gain proof of concept (POC). Here, we review preclinical animal models associated with development of new therapies for diseases of the ocular surface, glaucoma, presbyopia, and retinal diseases, including diabetic retinopathy and age-related macular degeneration (AMD). We have focused on summarizing the models critical to new drug development and described the translational features of the models that contributed to our understanding of disease pathogenesis and establishment of preclinical POC.

Review of Biomarkers in Ocular Matrices: Challenges and Opportunities.

Biomarkers provide a powerful and dynamic approach to improve our understanding of the mechanisms underlying ocular diseases with applications in diagnosis, disease modulation or for predicting and monitoring of clinical response to treatment. Defined as measurable indicator of normal or pathological processes, biomarker evaluation has been used extensively in drug development within clinical settings to better comprehend effectiveness of treatment in ocular diseases. Biomarkers in the eye have the advantage of access to multiple ocular matrices via minimally invasive methods. Repeat sampling for biomarker assessment has enabled reproducible objective measures of disease process or biological responses to a drug treatment. This review describes the usage of biomarkers with respect to four commonly sampled ocular matrices in clinic: tears, conjunctiva, aqueous humor and vitreous. Issues that affect the evaluation of biomarkers are discussed along with opportunities to leverage biomarkers such that ultimately, they can be used for customized targeted therapy.

Function of brain α2B-adrenergic receptor characterized with subtype-selective α2B antagonist and KO mice.

Noradrenergic signaling, through the α2A and α2C adrenergic receptors modulates the cognitive and behavioral symptoms of disorders such as schizophrenia, attention deficit hyperactivity disorder (ADHD), and addiction. However, it is unknown whether the α2B receptor has any significant role in CNS function. The present study elucidates the potential role of the α2B receptor in CNS function via the discovery and use of the first subtype-selective α2B antagonist (AGN-209419), and behavioral analyses of α-receptor knockout (KO) mice. Using AGN-209419 as radioligand, α2B receptor binding sites were identified within the olfactory bulb, cortex, thalamus, cerebellum, and striatum. Based on the observed expression patterns of α2 subtypes in the brain, we compared α2B KO, α2A KO and α2C KO mice behavioral phenotypes with their respective wild-type lines in anxiety (plus maze), compulsive (marble burying), and sensorimotor (prepulse inhibition) tasks. α2B KO mice exhibited increased marble burying and α2C KO mice exhibited an increased startle response to a pulse stimulus, but otherwise intact prepulse inhibition. To further explore compulsive behavior, we evaluated novelty-induced locomotor hyperactivity and found that α2B KO and α2C KO mice exhibited increased locomotion in the open field. Interestingly, when challenged with amphetamine, α2C KO mice increased activity at lower doses relative to either α2A KO or WT mice. However, α2B KO mice exhibited stereotypy at doses of amphetamine that were only locomotor stimulatory to all other genotypes. Following co-administration of AGN-209419 with low-dose amphetamine in WT mice, stereotypy was observed, mimicking the α2B KO phenotype. These findings suggest that the α2B receptor is involved in CNS behaviors associated with sensorimotor gating and compulsivity, and may be therapeutically relevant for disorders such as schizophrenia, ADHD, post-traumatic stress disorder, addiction, and obsessive compulsive disorder.

Carbonic anhydrases and mucosal vanilloid receptors help mediate the hyperemic response to luminal CO2 in rat duodenum.

BACKGROUND & AIMS: The duodenal mucosa is exposed to PCO(2) >200 mm Hg due to the luminal mixture of gastric acid with secreted bicarbonate, which augments mucosal protective mechanisms. We examined the hyperemic response to elevated luminal PCO(2) in the duodenum of anesthetized rats luminally exposed to high CO(2) saline to help elucidate luminal acid-sensing mechanisms. METHODS: Blood flow was measured by laser Doppler, and intracellular pH of epithelial cells by measured by ratio microimaging. The permeant carbonic anhydrase (CA) inhibitor methazolamide, relatively impermeant CA inhibitor benzolamide, vanilloid receptor antagonist capsazepine, or sodium-hydrogen exchanger 1 (NHE-1) inhibitor dimethyl amiloride were perfused with or without the high CO(2) solution. RESULTS: The high CO(2) solution increased duodenal blood flow, which was abolished by pretreatment with methazolamide or capsazepine or by dimethyl amiloride coperfusion. Sensory denervation with capsaicin also abolished the CO(2) effects. Benzolamide dose-dependently inhibited CO(2)-induced hyperemia and at 100 nmol/L inhibited CO(2)-induced intracellular acidification. The membrane-bound CA isoforms IV, IX, XII, and XIV and cytosolic CA II and the vanilloid receptor 1 (TRPV1) were expressed in duodenum and stomach. Dorsal root ganglion and nodose ganglion expressed all isoforms except for CA IX. CONCLUSIONS: The duodenal hyperemic response to luminal CO(2) is dependent on cytosolic and membrane-bound CA isoforms, NHE-1, and TRPV1. CO(2)-induced intracellular acidification was inhibited by selective extracellular CA inhibition, suggesting that CO(2) diffusion across the epithelial apical membrane is mediated by extracellular CA. NHE-1 activation preceding TRPV1 stimulation suggests that luminal CO(2) is sensed as H(+) in the subepithelium.