Formoterol alters chemokine expression and ameliorates pain behaviors after moderate spinal cord injury in female mice.

Secondary spinal cord injury (SCI) is characterized by increased cytokines and chemokines at the site of injury that have been associated with the development of neuropathic pain. Nearly 80% of SCI patients report suffering from chronic pain, which is poorly managed with available analgesics. While treatment with the FDA-approved ß2-adrenergic receptor agonist, formoterol, improves various aspects of recovery post-SCI in vivo, its effects on cytokines, chemokines and neuropathic pain remain unknown. Female mice were subjected to moderate (60 kdyn) or severe (80 kdyn) SCI followed by daily treatment with vehicle or formoterol (0.3 mg/kg, i.p.) beginning 8h after injury. The expression of pro-inflammatory cytokines/chemokines, such as IP-10, MIP-1a, MCP-1, BCA-1 and NF-κB, was increased in the injury site of vehicle-treated mice 24h post-SCI, which was ameliorated with formoterol treatment, regardless of injury severity. Thermal hyperalgesia and mechanical allodynia, as measured by Hargreaves infrared apparatus and von Frey filaments, respectively, were assessed prior to SCI and then weekly beginning 21 days post injury (DPI). While all injured mice exhibited decreased withdrawal latency following thermal stimulation compared to baseline, formoterol treatment reduced this response ~15% by 35 DPI. Vehicle-treated mice displayed significant mechanical allodynia, as evidenced by a 55% decrease in withdrawal threshold from baseline. In contrast, mice treated with formoterol maintained a consistent withdrawal time at all times tested. These data indicate that formoterol reduces inflammation post-SCI, likely contributing to mitigation of neuropathic pain, and further supporting the therapeutic potential of this treatment strategy. Significance Statement Chronic pain is a detrimental consequence of spinal cord injury (SCI). We show that treatment with the FDA-approved drug formoterol after SCI decreases injury site pro-inflammatory chemo/cytokines and alters markers of glial cell activation and infiltration. Additionally, formoterol treatment improves locomotor function and body composition, and decreases lesion volume. Finally, formoterol treatment decreased mechanical allodynia and thermal hyperalgesia post-SCI. These data are suggestive of the mechanism of formoterol-induced recovery, and further indicate its potential as a therapeutic strategy for SCI.

5-HT1F receptor agonism induces mitochondrial biogenesis and increases cellular function in brain microvascular endothelial cells.

Introduction: Vascular and mitochondrial dysfunction are well-established consequences of multiple central nervous system (CNS) disorders, including neurodegenerative diseases and traumatic injuries. We previously reported that 5-hydroxytryptamine 1F receptor (5-HT1FR) agonism induces mitochondrial biogenesis (MB) in multiple organ systems, including the CNS. Methods: Lasmiditan is a selective 5-HT1FR agonist that is FDA-approved for the treatment of migraines. We have recently shown that lasmiditan treatment induces MB, promotes vascular recovery and improves locomotor function in a mouse model of spinal cord injury (SCI). To investigate the mechanism of this effect, primary cerebral microvascular endothelial cells from C57bl/6 mice (mBMEC) were used. Results: Lasmiditan treatment increased the maximal oxygen consumption rate, mitochondrial proteins and mitochondrial density in mBMEC, indicative of MB induction. Lasmiditan also enhanced endothelial cell migration and tube formation, key components of angiogenesis. Trans-endothelial electrical resistance (TEER) and tight junction protein expression, including claudin-5, were also increased with lasmiditan, suggesting improved barrier function. Finally, lasmiditan treatment decreased phosphorylated VE-Cadherin and induced activation of the Akt-FoxO1 pathway, which decreases FoxO1-mediated inhibition of claudin-5 transcription. Discussion: These data demonstrate that lasmiditan induces MB and enhances endothelial cell function, likely via the VE-Cadherin-Akt-FoxO1-claudin-5 signaling axis. Given the importance of mitochondrial and vascular dysfunction in neuropathologies, 5-HT1FR agonism may have broad therapeutic potential to address multiple facets of disease progression by promoting MB and vascular recovery.

Isolation and monoculture of functional primary astrocytes from the adult mouse spinal cord.

Astrocytes are a widely heterogenic cell population that play major roles in central nervous system (CNS) homeostasis and neurotransmission, as well as in various neuropathologies, including spinal cord injury (SCI), traumatic brain injury, and neurodegenerative diseases, such as amyotrophic lateral sclerosis. Spinal cord astrocytes have distinct differences from those in the brain and accurate modeling of disease states is necessary for understanding disease progression and developing therapeutic interventions. Several limitations to modeling spinal cord astrocytes in vitro exist, including lack of commercially available adult-derived cells, lack of purchasable astrocytes with different genotypes, as well as time-consuming and costly in-house primary cell isolations that often result in low yield due to small tissue volume. To address these issues, we developed an efficient adult mouse spinal cord astrocyte isolation method that utilizes enzymatic digestion, debris filtration, and multiple ACSA-2 magnetic microbead purification cycles to achieve an astrocyte monoculture purity of ≅93-98%, based on all markers assessed. Importantly, the isolated cells contain active mitochondria and express key astrocyte markers including ACSA-1, ACSA-2, EAAT2, and GFAP. Furthermore, this isolation method can be applied to the spinal cord of male and female mice, mice subjected to SCI, and genetically modified mice. We present a primary adult mouse spinal cord astrocyte isolation protocol focused on purity, viability, and length of isolation that can be applied to a multitude of models and aid in targeted research on spinal-cord related CNS processes and pathologies.

A refined protocol for the isolation and monoculture of primary mouse renal peritubular endothelial cells.

During an episode of acute kidney injury (AKI), a sudden and rapid decline in renal function is often accompanied by a persistent reduction in mitochondrial function, microvasculature dysfunction/rarefaction, and tubular epithelial injury/necrosis. Additionally, patients who have experienced an AKI are at an elevated risk of developing other progressive renal, cardiovascular, and cardiorenal related diseases. While restoration of the microvasculature is imperative for oxygen and nutrient delivery/transport during proper renal repair processes, the mechanism(s) by which neovascularization and/or inhibition of microvascular dysfunction improves renal recovery remain understudied. Interestingly, pharmacological stimulation of mitochondrial biogenesis (MB) post-AKI has been shown to restore mitochondrial and renal function in mice. Thus, targeting MB pathways in microvasculature endothelial cell (MV-EC) may provide a novel strategy to improve renal vascular function and repair processes post-AKI. However, limitations to studying such mechanisms include a lack of commercially available primary renal peritubular MV-ECs, the variability in both purity and outgrowth of primary renal MV-EC in monoculture, the tendency of primary renal MV-ECs to undergo phenotypic loss in primary monoculture, and a limited quantity of published protocols to obtain primary renal peritubular MV-ECs. Thus, we focused on refining the isolation and phenotypic retention of mouse renal peritubular endothelial cells (MRPEC) for future physiological and pharmacological based studies. Here, we present a refined isolation method that augments the purity, outgrowth, and phenotypic retention of primary MRPEC monocultures by utilizing a collagenase type I enzymatic digestion, CD326+ (EPCAM) magnetic microbead epithelial cell depletion, and two CD146+ (MCAM) magnetic microbead purification cycles to achieve a monoculture MRPEC purity of ≅ 91-99% by all markers evaluated.

Increased Renal Expression of Complement Components in Patients With Liver Diseases: Nonalcoholic Steatohepatitis, Alcohol-Associated, Viral Hepatitis, and Alcohol-Viral Combination.

Inflammatory liver diseases, including nonalcoholic steatohepatitis (NASH), alcohol-associated liver disease (ALD), hepatitis C virus (HCV), and ALD/HCV, account for nearly 2 million deaths annually. Despite increasing evidence that liver dysfunction impacts renal physiology, there is limited supportive clinical information, due to limited diagnosis of liver disease, complexity in liver disease etiology, and inadequacy of renal function tests. Human kidney biopsies with liver and renal pathology were obtained from patients with nonalcoholic fatty liver disease (NAFLD), NASH, ALD, HCV, and ALD/HCV (n = 5-7). Each liver disease showed renal pathology with at least 50% interstitial nephritis, 50% interstitial fibrosis, and renal dysfunction by estimated glomerular filtration rate (NAFLD 36.7 ± 21.4; NASH 32.7 ± 15.0; ALD 16.0 ± 11.0; HCV 27.6 ± 11.5; ALD/HCV 21.0 ± 11.2 ml/min/1.73 m2). Transcriptomic analysis identified 55 genes with expression changes in a conserved direction in response to liver disease. Considering association with immune regulation, protein levels of alpha-2-macroglobulin, clusterin, complement C1q C chain (C1QC), CD163, and joining chain of multimeric IgA and IgM (JCHAIN) were further quantified by LC-MS/MS. C1QC demonstrated an increase in NASH, ALD, HCV, and ALD/HCV (42.9 ± 16.6; 38.8 ± 18.4; 39.0 ± 13.5; 40.1 ± 20.1 pmol/mg protein) relative to control (19.2 ± 10.4 pmol/mg protein; p ≤ 0.08). Renal expression changes identified in inflammatory liver diseases with interstitial pathology suggest the pathogenesis of liver associated renal dysfunction. This unique cohort overcomes diagnostic discrepancies and sample availability to provide insight for mechanistic investigations on the impact of liver dysfunction on renal physiology.

Baseline effects of lysophosphatidylcholine and nerve growth factor in a rat model of sciatic nerve regeneration after crush injury.

Schwann cells play a major role in helping heal injured nerves. They help clear debris, produce neurotrophins, upregulate neurotrophin receptors, and form bands of Büngner to guide the healing nerve. But nerves do not always produce enough neurotrophins and neurotrophin receptors to repair themselves. Nerve growth factor (NGF) is an important neurotrophin for promoting nerve healing and lysophosphatidylcholine (LPC) has been shown to stimulate NGF receptors (NGFR). This study tested the administration of a single intraneural injection of LPC (1 mg/mL for single LPC injection and 10 mg/mL for multiple LPC injections) at day 0 and one (day 7), two (days 5 and 7), or three (days 5, 7, and 9) injections of NGF (160 ng/mL for single injections and 80 ng/mL for multiple injections) to determine baseline effects on crushed sciatic nerves in rats. The rats were randomly divided into four groups: control, crush, crush-NGF, and crush-LPC-NGF. The healing of the nerves was measured weekly by monitoring gait; electrophysiological parameters: compound muscle action potential (CMAP) amplitudes; and morphological parameters: total fascicle areas, myelinated fiber counts, fiber densities, fiber packing, and mean g-ratio values at weeks 3 and 6. The crush, crush-NGF, and crush-LPC-NGF groups statistically differed from the control group for all six weeks for the electrophysiological parameters but only differed from the control group at week 3 for the morphological parameters. The crush, crush-NGF, and crush-LPC-NGF groups did not differ from each other over the course of the study. Single injections of LPC and NGF one week apart or multiple treatments of NGF at 5, 7 and 9 days post-injury did not alter the healing rate of the sciatic nerves during weeks 1-6 of the study. These findings are important to define the baseline effects of NGF and LPC injections, as part of a larger effort to determine the minimal dose regimen of NGF to regenerate peripheral nerves.