Browsing by Author "Brooks, Calvin D."
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Item Effect of systemic administration of α7-nicotinic acetylcholine receptor ligands on renal inflammation in young mice with systemic lupus erythematosus(2022) Brooks, Calvin D.; Young-Stubbs, Cassandra M.; Shimoura, Caroline; Dinh, Viet; Chaudhari, Sarika; Uteshev, Victor; Mathis, Keisa W.Systemic lupus erythematosus (SLE) is an autoimmune disease where renal inflammation contributes to hypertension. The cholinergic anti-inflammatory pathway is a recently described pathway where stimulating the vagus nerve causes release of acetylcholine from choline acetyltransferase (ChAT)+ T-cells in the spleen. This acetylcholine acts on alpha-7 nicotinic acetylcholine receptors (α7nAChR) of immune cells to hault the production of pro-inflammatory cytokines. Our lab has shown stimulation of this pathway at multiple levels lessens autoimmunity, renal inflammation and hypertension in SLE mice. However, our recent attempts to target the α7nAChR directly with a positive allosteric modulator (PAM) in mice with advanced SLE have not yielded similar results. This may be due to decreased parasympathetic tone in these mice in which the PAM is not able to compensate for. The aim of the current study was to determine if activating the α7nAChR in SLE mice at an earlier age, before dampening of parasympathetic tone, prevents the onset of hypertension and renal inflammation. Twelve week old female NZBWF1 mice, which spontaneously develop SLE, and NZW controls were given a partial agonist of the α7nAChR, GTS-21, a PAM, PNU-120596, or vehicle continuously for two weeks via subcutaneous osmotic mini-pump. Mean arterial pressure (MAP) was measured by carotid artery catheter in conscious, freely moving mice at 14 weeks. Mice were then euthanized and blood, spleen and kidneys harvested to allow measurement of plasma double stranded (ds) DNA autoantibodies via ELISA to assess severity of disease. There was no difference in dsDNA autoantibody activity (U/mL) between SLE mice and controls (all data presented as mean±SEM; 76026.3±38901.4 vs. 19617.4±4092.7; p=0.1141). The treatments had no effect on autoantibody activity in SLE mice [76026.3±38901.4 (SLE vehicle) vs. 36951.7±5962.3 (SLE PNU) vs. 56279.7±31381.0 (SLE GTS)] or controls [19617.4±4092.7 (Con vehicle) vs. 17293.2±3384.1 (Con PNU) vs. 16016.2±3059.6 (Con GTS)]. MAP (mmHg) did not differ significantly between young SLE and control mice (143.53±3.26 vs. 128.8±4.95). Additionally, the treatments had no effect on MAP of SLE mice [143.53±3.26 (SLE vehicle) vs. 128.32±10.92 (SLE PNU) vs. 129.56±19.50 (SLE GTS)] or controls [128.8±4.95 (Con vehicle) vs. 127.60±4.43 (Con PNU), vs. 125.65±5.54 (Con GTS)]. Based on these results, we suspect that the disease process has not progressed enough in 14-week-old mice to see differences due to these treatments. Although the changes in the blood pressure and dsDNA antibodies are not significant, we will continue to evaluate renal damage and cytokine profile to determine the effect of these α7nAChR ligands on pathogenesis of SLE. Future studies will aim to modulate α7nAChRs in SLE mice before the onset of disease (~12 weeks of age) through 35 weeks when mice usually experience terminal disease to determine efficacy of early activation of the cholinergic anti-inflammatory pathway in halting the progression of SLE.Item Intravitreal Endothelin-1 (ET-1) Injection Reduces Mitophagy in Retinal Ganglion Cells in MitoQC Mice(2023) Brooks, Calvin D.; Kodati, Bindu; Inman, Denise; Stankowska, Dorota; Krishnamoorthy, RaghuPurpose: The peptide endothelin-1 (ET-1), and its receptors are upregulated in the aqueous humor and retina in animal models of experimentally induced ocular hypertension, and have been shown to have a causative role in retinal ganglion cell (RGC) neurodegeneration. The purpose of this experiment was to assess the role of mitophagy in RGC neurodegeneration following intravitreal ET-1 administration in MitoQC mice. Methods: MitoQC mice (Gt(ROSA)26Sortm1(CAG-mCherry/GFP)Ganl on a C57BL/6 background) at the age of 3 months were used for the study. The mitochondria in these mice display both red and green fluorescence due to expression of a mCherry-GFP tag fused to the mitochondrial targeting sequence of an outer mitochondrial membrane protein, FIS1. When these mitochondria are trafficked to the lysosome for degradation, the green fluorescence is quenched, leaving only the red fluorescence. The MitoQC mice were intravitreally injected in both eyes with either ET-1 (1 nmole) or vehicle (water), and 72 hours following the injections the mice eyes were enucleated and retinal flat mounts were live-imaged using a Zeiss LSM 880 super resolution confocal microscope. Z-stack imaging was used to image the ganglion cell layer. For each Z layer, a threshold algorithm was used to define a region of interest (ROI) that included only areas with red fluorescence, after which red and green fluorescence were quantified for that ROI. Red/green fluorescence intensity was calculated and averaged per image. A red/green ratio larger than 1 is indicative of active mitophagy. This ratio was compared between ET-1 and vehicle-injected mice using a Mann-Whitney test (n=4 eyes per group). Results: At 72 hours after injection with ET-1, the average red/green fluorescence ratio in the RGCs was 0.86, while the vehicle-injected mice had an average red/green ratio of 1.29. These ratios were significantly different (P=0.0003), and the smaller red/green ratio in the ET-1 group indicates lesser mitophagy than the vehicle group. Conclusion: Mitophagy is known to be an important quality control mechanism for neuronal cell survival, and this study provides evidence that mitophagy is impaired by ET-1. The finding indicates that a decline in mitophagy may be associated with endothelin-mediated neurodegeneration in RGCs.Item Role of mitophagy in ocular neurodegeneration(Frontiers Media S.A., 2023-11-15) Brooks, Calvin D.; Kodati, Bindu; Stankowska, Dorota L.; Krishnamoorthy, Raghu R.Neurons in the central nervous system are among the most metabolically active cells in the body, characterized by high oxygen consumption utilizing glucose both aerobically and anaerobically. Neurons have an abundance of mitochondria which generate adequate ATP to keep up with the high metabolic demand. One consequence of the oxidative phosphorylation mechanism of ATP synthesis, is the generation of reactive oxygen species which produces cellular injury as well as damage to mitochondria. Mitochondria respond to injury by fusion which serves to ameliorate the damage through genetic complementation. Mitochondria also undergo fission to meet an increased energy demand. Loss of mitochondria is also compensated by increased biogenesis to generate new mitochondria. Damaged mitochondria are removed by mitophagy, an autophagic process, in which damaged mitochondria are surrounded by a membrane to form an autophagosome which ultimately fuses with the lysosome resulting in degradation of faulty mitochondria. Dysregulation of mitophagy has been reported in several central nervous system disorders, including, Alzheimer's disease and Parkinson's disease. Recent studies point to aberrant mitophagy in ocular neurodegenerative disorders which could be an important contributor to the disease etiology/pathology. This review article highlights some of the recent findings that point to dysregulation of mitophagy and it's underlying mechanisms in ocular neurodegenerative diseases, including, glaucoma, age-related macular degeneration and diabetic retinopathy.Item Should Renal Inflammation Be Targeted While Treating Hypertension?(Frontiers Media S.A., 2022-06-13) Chaudhari, Sarika; Pham, Grace S.; Brooks, Calvin D.; Dinh, Viet Q.; Young-Stubbs, Cassandra M.; Shimoura, Caroline G.; Mathis, Keisa W.Despite extensive research and a plethora of therapeutic options, hypertension continues to be a global burden. Understanding of the pathological roles of known and underexplored cellular and molecular pathways in the development and maintenance of hypertension is critical to advance the field. Immune system overactivation and inflammation in the kidneys are proposed alternative mechanisms of hypertension, and resistant hypertension. Consideration of the pathophysiology of hypertension in chronic inflammatory conditions such as autoimmune diseases, in which patients present with autoimmune-mediated kidney inflammation as well as hypertension, may reveal possible contributors and novel therapeutic targets. In this review, we 1) summarize current therapies used to control blood pressure and their known effects on inflammation; 2) provide evidence on the need to target renal inflammation, specifically, and especially when first-line and combinatory treatment efforts fail; and 3) discuss the efficacy of therapies used to treat autoimmune diseases with a hypertension/renal component. We aim to elucidate the potential of targeting renal inflammation in certain subsets of patients resistant to current therapies.