Cytokine Release by Microglia Exposed to Neurologic Injury Is Amplified by Lipopolysaccharide.

INTRODUCTION: Traumatic brain injury (TBI) is a leading cause of death and morbidity in the trauma population. Microglia drive the secondary neuroinflammatory response after TBI. We sought to determine if the microglial response to neurologic injury was exacerbated by a second stimulus after exposure to neurologic injury. METHODS: Sprague-Dawley rats (age 2-3 wk) were divided into injured and noninjured groups. Injured rats underwent a controlled cortical impact injury; noninjured rats remained naïve to any injury and served as the control group. Primary rat microglia were isolated and applied to in vitro cultures. After incubation for 24 h, the microglia were stimulated with lipopolysaccharide (LPS) or norepinephrine. Twenty-four hours after stimulation, cell culture supernatant was collected. Tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) production were measured by standard enzyme-linked immunosorbent assays. GraphPad Prism was used for statistical analysis. RESULTS: When compared to noninjured microglia, LPS induced a significantly greater production of TNF-α in microglia isolated from the injured ipsilateral (versus noninjured = 938.8 ± 155.1, P < 0.0001) and injured contralateral hemispheres (versus noninjured = 426.6 ± 155.1, P < 0.0001). When compared to microglia from noninjured cerebral tissue, IL-6 production was significantly greater after LPS stimulation in the injured ipsilateral hemisphere (mean difference versus noninjured = 9540 ± 3016, P = 0.0101) and the contralateral hemisphere (16,700 ± 3016, P < 0.0001). Norepinephrine did not have a significant effect on IL-6 or TNF-α production. CONCLUSIONS: LPS stimulation may amplify the release of proinflammatory cytokines from postinjury microglia. These data suggest that post-TBI complications, like sepsis, may propagate neuroinflammation by augmenting the proinflammatory response of microglia.

Assessment of spinal cord injury using ultrasound elastography in a rabbit model in vivo.

The effect of the mechanical micro-environment on spinal cord injury (SCI) and treatment effectiveness remains unclear. Currently, there are limited imaging methods that can directly assess the localized mechanical behavior of spinal cords in vivo. In this study, we apply new ultrasound elastography (USE) techniques to assess SCI in vivo at the site of the injury and at the time of one week post injury, in a rabbit animal model. Eleven rabbits underwent laminectomy procedures. Among them, spinal cords of five rabbits were injured during the procedure. The other six rabbits were used as control. Two neurological statuses were achieved: non-paralysis and paralysis. Ultrasound data were collected one week post-surgery and processed to compute strain ratios. Histologic analysis, mechanical testing, magnetic resonance imaging (MRI), computerized tomography and MRI diffusion tensor imaging (DTI) were performed to validate USE results. Strain ratios computed via USE were found to be significantly different in paralyzed versus non-paralyzed rabbits. The myelomalacia histologic score and spinal cord Young's modulus evaluated in selected animals were in good qualitative agreement with USE assessment. It is feasible to use USE to assess changes in the spinal cord of the presented animal model. In the future, with more experimental data available, USE may provide new quantitative tools for improving SCI diagnosis and prognosis.

Innate immune activation and white matter injury in a rat model of neonatal intraventricular hemorrhage are dependent on developmental stage.

BACKGROUND: Inflammation and white matter injury are consequences of neonatal intraventricular hemorrhage (IVH). Both white matter and the neuroimmune system are developing during the time which IVH occurs and its consequences develop. IVH has been studied in many different animal models; however, the effects of IVH occurring at different developmental time points in the same model have not been examined. Understanding how the timing of IVH affects outcome may provide important insights into both IVH pathophysiology and innate immune development. METHODS: We used intraventricular injection of lysed whole blood to model neonatal IVH in postnatal day (P)2 and P5 rats. Flow cytometry was used to detect innate immune activation. MRI was used to screen animals for the development of increased ventricular size. Immunohistochemistry for myelin basic protein was used to quantify white matter and corpus callosum thickness. RESULTS: P5 animals exhibited significant increases in several measures of classically pro-inflammatory innate immune activation that P2 animals did not. Animals with IVH induced at P5 also developed ventricular enlargement visible on MRI whereas animals with IVH induced at P2 did not. On histological analysis, there were no significant effects of IVH in P2 animals, but IVH in P5 animals reduced white matter labeling and corpus callosum thickness. CONCLUSIONS: IVH induces a strong innate inflammatory response in P5 as well as changes in ventricular size and reduction of white matter. P2 animals do not exhibit significant changes in innate immune activation or white matter structure after IVH. This suggests that white matter pathology from IVH is due in part to innate immune activation; and that the developmental stage of the innate immune system is a key determinant of IVH pathology.

Innate immune activation and white matter injury in a rat model of neonatal intraventricular hemorrhage are dependent on developmental stage.

Background: Inflammation and white matter injury are consequences of neonatal intraventricular hemorrhage (IVH). Both white matter and the neuroimmune system are developing during which IVH and its consequences occur. IVH has been studied in many different animal models; however, the effects of IVH occurring at different developmental time points in the same model has not been examined. Examining how the timing of IVH affects the ultimate outcome of IVH may provide important insights into IVH pathophysiology. Methods: We used intraventricular injection of lysed whole blood to model neonatal IVH in postnatal day (P)2 and P5 rats. Flow cytometry was used to detect innate immune activation. MRI was used to screen animals for the development of increased ventricular size. Immunohistochemistry for myelin basic protein was used to assess white matter pathology. Results: The acute response of the innate immune system at these time points differed, with P5 animals exhibiting significant increases in several measures of classically pro-inflammatory innate immune activation that P2 animals did not. Animals with IVH induced at P5 also developed ventricular enlargement visible on MRI whereas animals with IVH induced at P2 did not. On histological analysis, there were no significant effects of IVH in P2 animals, but IVH in P5 animals induced a reduction in several measures of white matter integrity. Conclusions: IVH induces a strong innate inflammatory response in P5 animals that correlates with changes in ventricular size and white matter. P2 animals did not exhibit any significant changes in innate immune activation or white matter structure after IVH. This suggests that the white matter pathology from IVH is due in part to innate immune activation; and that the developmental stage of the innate immune system is a key determinant of IVH pathology.

Dexmedetomidine Alters the Inflammatory Profile of Rat Microglia In Vitro.

BACKGROUND: Microglia are a primary mediator of the neuroinflammatory response to neurologic injury, such as that in traumatic brain injury. Their response includes changes to their cytokine expression, metabolic profile, and immunophenotype. Dexmedetomidine (DEX) is an α2 adrenergic agonist used as a sedative in critically ill patients, such as those with traumatic brain injury. Given its pharmacologic properties, DEX may alter the phenotype of inflammatory microglia. METHODS: Primary microglia were isolated from Sprague-Dawley rats and cultured. Microglia were activated using multiple mediators: lipopolysaccharide (LPS), polyinosinic-polycytidylic acid (Poly I:C), and traumatic brain injury damage-associated molecular patterns (DAMP) from a rat that sustained a prior controlled cortical impact injury. After activation, cultures were treated with DEX. At the 24-h interval, the cell supernatant and cells were collected for the following studies: cytokine expression (tumor necrosis factor-α [TNFα], interleukin-10 [IL-10]) via enzyme-linked immunosorbent assay, 6-phosphofructokinase enzyme activity assay, and immunophenotype profiling with flow cytometry. Cytokine expression and metabolic enzyme activity data were analyzed using two-way analysis of variance. Cell surface marker expression was analyzed using FlowJo software. RESULTS: In LPS-treated cultures, DEX treatment decreased the expression of TNFα from microglia (mean difference = 121.5 ± 15.96 pg/mL; p < 0.0001). Overall, DEX-treated cultures had a lower expression of IL-10 than nontreated cultures (mean difference = 39.33 ± 14.50 pg/mL, p < 0.0001). DEX decreased IL-10 expression in LPS-stimulated microglia (mean difference = 74.93 ± 12.50 pg/mL, p = 0.0039) and Poly I:C-stimulated microglia (mean difference = 23.27 ± 6.405 pg/mL, p = 0.0221). In DAMP-stimulated microglia, DEX decreased the activity of 6-phosphofructokinase (mean difference = 18.79 ± 6.508 units/mL; p = 0.0421). The microglial immunophenotype was altered to varying degrees with different inflammatory stimuli and DEX treatment. CONCLUSIONS: DEX may alter the neuroinflammatory response of microglia. By altering the microglial profile, DEX may affect the progression of neurologic injury.

An Enzymatically Cleavable Tripeptide Linker for Maximizing the Therapeutic Index of Antibody-Drug Conjugates.

Valine-citrulline is a protease-cleavable linker commonly used in many drug delivery systems, including antibody-drug conjugates (ADC) for cancer therapy. However, its suboptimal in vivo stability can cause various adverse effects such as neutropenia and hepatotoxicity, leading to dose delays or treatment discontinuation. Here, we report that glutamic acid-glycine-citrulline (EGCit) linkers have the potential to solve this clinical issue without compromising the ability of traceless drug release and ADC therapeutic efficacy. We demonstrate that our EGCit ADC resists neutrophil protease-mediated degradation and spares differentiating human neutrophils. Notably, our anti-HER2 ADC shows almost no sign of blood and liver toxicity in healthy mice at 80 mg kg-1. In contrast, at the same dose level, the FDA-approved anti-HER2 ADCs Kadcyla and Enhertu show increased levels of serum alanine aminotransferase and aspartate aminotransferase and morphologic changes in liver tissues. Our EGCit conjugates also exert greater antitumor efficacy in multiple xenograft tumor models compared with Kadcyla and Enhertu. This linker technology could substantially broaden the therapeutic windows of ADCs and other drug delivery agents, providing clinical options with improved efficacy and safety.

Cyclic stretch-induced stress fiber dynamics - dependence on strain rate, Rho-kinase and MLCK.

Stress fiber realignment is an important adaptive response to cyclic stretch for nonmuscle cells, but the mechanism by which such reorganization occurs is not known. By analyzing stress fiber dynamics using live cell microscopy, we revealed that stress fiber reorientation perpendicular to the direction of cyclic uniaxial stretching at 1 Hz did not involve disassembly of the stress fiber distal ends located at focal adhesion sites. Instead, these distal ends were often used to assemble new stress fibers oriented progressively further away from the direction of stretch. Stress fiber disassembly and reorientation were not induced when the frequency of stretch was decreased to 0.01 Hz, however. Treatment with the Rho-kinase inhibitor Y27632 reduced stress fibers to thin fibers located in the cell periphery which bundled together to form thick fibers oriented parallel to the direction of stretching at 1Hz. In contrast, these thin fibers remained diffuse in cells subjected to stretch at 0.01 Hz. Cyclic stretch at 1 Hz also induced actin fiber formation parallel to the direction of stretch in cells treated with the myosin light chain kinase (MLCK) inhibitor ML-7, but these fibers were located centrally rather than peripherally. These results shed new light on the mechanism by which stress fibers reorient in response to cyclic stretch in different regions of the actin cytoskeleton.