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.

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.

Non-cytotoxic Dityrosine Photocrosslinked Polymeric Materials With Targeted Elastic Moduli.

Controlling mechanical properties of polymeric biomaterials, including the elastic modulus, is critical to direct cell behavior, such as proliferation and differentiation. Dityrosine photocrosslinking is an attractive and simple method to prepare materials that exhibit a wide range of elastic moduli by rapidly crosslinking tyrosyl-containing polymers. However, high concentrations of commonly used oxidative crosslinking reagents, such as ruthenium-based photoinitiators and persulfates, present cytotoxicity concerns. We found the elastic moduli of materials prepared by crosslinking an artificial protein with tightly controlled tyrosine molarity can be modulated up to 40 kPa by adjusting photoinitiator and persulfate concentrations. Formulations with various concentrations of the crosslinking reagents were able to target a similar material elastic modulus, but excess unreacted persulfate resulted in cytotoxic materials. Therefore, we identified a systematic method to prepare non-cytotoxic photocrosslinked polymeric materials with targeted elastic moduli for potential biomaterials applications in diverse fields, including tissue engineering and 3D bioprinting.