Evaluating the comorbidities of age and cigarette smoking on stroke outcomes in the context of anti-complement mitigation strategies.

Multiple neuroprotective agents have shown beneficial effects in rodent models of stroke, but they have failed to translate in the clinic. In this perspective, we consider that a likely explanation for this failure, at least in part, is that there has been inadequate assessment of functional outcomes in preclinical stroke models, as well the use of young healthy animals that are not representative of clinical cohorts. Although the impact of older age and cigarette smoking comorbidities on stroke outcomes is well documented clinically, the impact of these (and other) stroke comorbidities on the neuroinflammatory response after stroke, as well as the response to neuroprotective agents, remains largely unexplored. We have shown that a complement inhibitor (B4Crry), that targets specifically to the ischemic penumbra and inhibits complement activation, reduces neuroinflammation and improves outcomes following murine ischemic stroke. For this perspective, we discuss the impact of age and smoking comorbidities on outcomes after stroke, and we experimentally assess whether increased complement activation contributes to worsened acute outcomes with these comorbidities. We found that the pro-inflammatory effects of aging and smoking contribute to worse stroke outcomes, and these effects are mitigated by complement inhibition.

A Role of Complement in the Pathogenic Sequelae of Mouse Neonatal Germinal Matrix Hemorrhage.

Germinal matrix hemorrhage (GMH) is a devastating disease of infancy that results in intraventricular hemorrhage, post-hemorrhagic hydrocephalus (PHH), periventricular leukomalacia, and neurocognitive deficits. There are no curative treatments and limited surgical options. We developed and characterized a mouse model of GMH based on the injection of collagenase into the subventricular zone of post-natal pups and utilized the model to investigate the role of complement in PHH development. The site-targeted complement inhibitor CR2Crry, which binds deposited C3 complement activation products, localized specifically in the brain following its systemic administration after GMH. Compared to vehicle, CR2Crry treatment reduced PHH and lesion size, which was accompanied by decreased perilesional complement deposition, decreased astrocytosis and microgliosis, and the preservation of dendritic and neuronal density. Complement inhibition also improved survival and weight gain, and it improved motor performance and cognitive outcomes measured in adolescence. The progression to PHH, neuronal loss, and associated behavioral deficits was linked to the microglial phagocytosis of complement opsonized neurons, which was reversed with CR2Crry treatment. Thus, complement plays an important role in the pathological sequelae of GMH, and complement inhibition represents a novel therapeutic approach to reduce the disease progression of a condition for which there is currently no treatment outside of surgical intervention.

State of the science in inflammation and stroke recovery: A systematic review.

Stroke is a major cause of mortality worldwide, and survivors often have major life-changing disabilities. Annually in the United States, an estimated 795,000 people experience a new or recurrent stroke. All types of stroke involve an inflammatory reaction that follows the initial phase of incidence. However, investigations into any links between inflammatory markers and recovery processes in the context of post-stroke rehabilitation are lacking. In this systematic review, we searched the literature in PubMed, SCOPUS, and CINAHL databases to gather information on inflammatory biomarkers related to stroke and their association with rehabilitation outcomes, according to PRISMA guidelines. Eleven articles (n=1.773 stroke patients) were selected. Immune markers (interleukin 6 [IL-6], C-reactive protein, IL-1α, tumor necrosis factor α, soluble intercellular adhesion molecule 1) and functional status assessments (Modified Rankin Score, National Institutes of Health Stroke Scale, Functional Independence Measure, etc.) were the primary measures used in the reviewed studies. We found preliminary evidence for the evaluation of inflammatory biomarkers post-stroke, including the role of inflammation in functional recovery and the influence of rehabilitation on inflammation. This is the first systematic review of the topic. The review identifies several gaps in the literature that are critical for understanding the potential use of inflammatory markers to improve post-stroke outcomes.

Mechanical activation of the angiotensin II type 1 receptor contributes to abdominal aortic aneurysm formation.

OBJECTIVE: The angiotensin II type 1 receptor (AT1R) can be activated under conditions of mechanical stretch in some cellular systems. Whether this activity influences signaling within the abdominal aorta to promote to abdominal aortic aneurysm (AAA) development remains unknown. We evaluated the hypothesis that mechanical AT1R activation can occur under conditions of hypertension (HTN) and contribute to AAA formation. METHODS: BPH/2 mice, which demonstrate spontaneous neurogenic, low-renin HTN, and normotensive BPN/3 mice underwent AAA induction via the calcium chloride model, with or without an osmotic minipump delivering 30 mg/kg/d of the AT1R blocker Losartan. Systolic blood pressure (SBP) was measured at baseline and weekly via a tail cuff. The aortic diameter (AoD) was measured at baseline and terminal surgery at 21 days by digital microscopy. Aortic tissue was harvested for immunoblotting (phosphorylated extracellular signal-regulated kinase-1 and -2 [pERK1/2] to ERK1/2 ratio) and expressed as the fold-change from the BPN/3 control mice. Aortic vascular smooth muscle cells (VSMCs) underwent stretch with or without Losartan (1 µM) treatment to assess the mechanical stimulation of ERK1/2 activity. Statistical analysis of the blood pressure, AoD, and VSMC ERK1/2 activity was performed using analysis of variance. However, the data distribution was determined to be log-normal (Shapiro-Wilk test) for ERK1/2 activity. Therefore, it was logarithmically transformed before analysis of variance. RESULTS: At baseline, the SBP was elevated in the BPH/2 mice relative to the BPN/3 mice (P < .05). Losartan treatment significantly reduced the SBP in both mouse strains (P < .05). AAA induction did not affect the SBP. At 21 days after induction, the percentage of increase in the AoD from baseline was significantly greater in the BPH/2 mice than in the BPN/3 mice (101.28% ± 4.19% vs 75.59% ± 1.67% above baseline; P < .05). Losartan treatment significantly attenuated AAA growth in both BPH/2 and BPN/3 mice (33.88% ± 2.97% and 43.96% ± 3.05% above baseline, respectively; P < .05). ERK1/2 activity was increased approximately fivefold in the BPH/2 control mice relative to the BPN/3 control mice (P < .05). In the BPH/2 and BPN/3 mice with AAA, ERK1/2 activity was significantly increased relative to the respective baseline control (P < .05) and effectively reduced by concomitant Losartan therapy (P < .05). Biaxial stretch of the VSMCs in the absence of angiotensin II demonstrated increased ERK1/2 activation (P < .05 vs static control), which was significantly inhibited by Losartan. CONCLUSIONS: In BPH/2 mice with spontaneous neurogenic, low-renin HTN, AAA growth was amplified compared with the normotensive control and was effectively attenuated using Losartan. ERK1/2 activity was significantly elevated in the BPH/2 mice and after AAA induction in the normotensive and hypertensive mice but was attenuated by Losartan treatment. These data suggest that AT1R activation contributes to AAA development. Therefore, further investigation into this signaling pathway could establish targets for pharmacotherapeutic engineering to slow AAA growth. (JVS-Vascular Science 2021;2:194-206.). CLINICAL RELEVANCE: Hypertension (HTN) and abdominal aortic aneurysm (AAA) have been epidemiologically linked for decades; however, a biomechanical link has not yet been identified. Using a murine model of spontaneous neurogenic HTN experimentally demonstrated to have low circulating renin, mechanical activation of the angiotensin II type 1 receptor (AT1R) was identified with elevated blood pressure and AAA induction. HTN amplified AAA growth. However, more importantly, blocking the activation of AT1R with the angiotensin receptor blocker Losartan effectively abrogated AAA development. Although inhibiting the production of angiotensin II has previously been unsuccessful in altering AAA growth, the results from the present study suggest that blocking the activation of AT1R through direct ligand binding or mechanical stimulation might alter aortic wall signaling and warrants further investigation.

Complement mediates neuroinflammation and cognitive decline at extended chronic time points after traumatic brain injury.

Traumatic brain injury (TBI) can result in progressive cognitive decline occurring for years after the initial insult, and for which there is currently no pharmacological treatment. An ongoing chronic inflammatory response after TBI is thought to be an important factor in driving this cognitive decline. Here, we investigate the role of complement in neuroinflammation and cognitive decline for up to 6 months after murine TBI. Male C57BL/6 mice were subjected to open head injury using a controlled cortical impact device. At 2 months post TBI, mice were moved to large cages with an enriched environment to simulate rehabilitation therapy, and assigned to one of three treatment groups: 1. vehicle (PBS), 2. CR2Crry (3 doses over 1 week), 3. CR2Crry (continuous weekly dose until the end of the study). The study was terminated at 6 months post-TBI for all groups. Motor and cognitive function was analyzed, with histopathological analysis of brain tissue. Measured at 6 months after TBI, neither of the complement inhibition paradigms improved motor performance. However, mice receiving continuous CR2Crry treatment showed improved spatial learning and memory compared to both mice receiving only 3 doses and to mice receiving vehicle control. Analysis of brain sections at 6 months after injury revealed ongoing complement activation in the control group, with reduced complement activation and C3 deposition in the continuous CR2Crry treatment group. The ipsilateral hemisphere of continuously treated animals also showed a decrease in microglia/macrophage and astrocyte activation compared to vehicle. There was also increased astrocytosis in the contralateral hippocampus of vehicle treated vs. naïve mice, which was reduced in mice continuously treated with CR2Crry. This study demonstrates continued complement mediated neuroinflammation at extended chronic time points after TBI, and extends the potential treatment window for complement inhibition, which has previously been shown to improve outcomes after murine TBI.

Complement Drives Synaptic Degeneration and Progressive Cognitive Decline in the Chronic Phase after Traumatic Brain Injury.

Cognitive deficits following traumatic brain injury (TBI) remain a major cause of disability and early-onset dementia, and there is increasing evidence that chronic neuroinflammation occurring after TBI plays an important role in this process. However, little is known about the molecular mechanisms responsible for triggering and maintaining chronic inflammation after TBI. Here, we identify complement, and specifically complement-mediated microglial phagocytosis of synapses, as a pathophysiological link between acute insult and a chronic neurodegenerative response that is associated with cognitive decline. Three months after an initial insult, there is ongoing complement activation in the injured brain of male C57BL/6 mice, which drives a robust chronic neuroinflammatory response extending to both hemispheres. This chronic neuroinflammatory response promotes synaptic degeneration and predicts progressive cognitive decline. Synaptic degeneration was driven by microglial phagocytosis of complement-opsonized synapses in both the ipsilateral and contralateral brain, and complement inhibition interrupted the degenerative neuroinflammatory response and reversed cognitive decline, even when therapy was delayed until 2 months after TBI. These findings provide new insight into our understanding of TBI pathology and its management; and whereas previous therapeutic investigations have focused almost exclusively on acute treatments, we show that all phases of TBI, including at chronic time points after TBI, may be amenable to therapeutic interventions, and specifically to complement inhibition.SIGNIFICANCE STATEMENT There is increasing evidence of a chronic neuroinflammatory response after traumatic brain injury (TBI), but little is known about the molecular mechanisms responsible for triggering and maintaining chronic inflammation. We identify complement, and specifically complement-mediated microglial phagocytosis of synapses, as a pathophysiological link between acute insult and a chronic neurodegenerative response, and further that this response is associated with cognitive decline. Complement inhibition interrupted this response and reversed cognitive decline, even when therapy was delayed until 2 months after injury. The data further support the concept that TBI should be considered a chronic rather than an acute disease condition, and have implications for the management of TBI in the chronic phase of injury, specifically with regard to the therapeutic application of complement inhibition.

Anti-inflammatory and Neuroprotective Agents in Clinical Trials for CNS Disease and Injury: Where Do We Go From Here?

Neurological disorders are major contributors to death and disability worldwide. The pathology of injuries and disease processes includes a cascade of events that often involve molecular and cellular components of the immune system and their interaction with cells and structures within the central nervous system. Because of this, there has been great interest in developing neuroprotective therapeutic approaches that target neuroinflammatory pathways. Several neuroprotective anti-inflammatory agents have been investigated in clinical trials for a variety of neurological diseases and injuries, but to date the results from the great majority of these trials has been disappointing. There nevertheless remains great interest in the development of neuroprotective strategies in this arena. With this in mind, the complement system is being increasingly discussed as an attractive therapeutic target for treating brain injury and neurodegenerative conditions, due to emerging data supporting a pivotal role for complement in promoting multiple downstream activities that promote neuroinflammation and degeneration. As we move forward in testing additional neuroprotective and immune-modulating agents, we believe it will be useful to review past trials and discuss potential factors that may have contributed to failure, which will assist with future agent selection and trial design, including for complement inhibitors. In this context, we also discuss inhibition of the complement system as a potential neuroprotective strategy for neuropathologies of the central nervous system.

Complement-Mediated Microglial Phagocytosis and Pathological Changes in the Development and Degeneration of the Visual System.

The focus of this review is the role of complement-mediated phagocytosis in retinal and neurological diseases affecting the visual system. Complement activation products opsonize synaptic material on neurons for phagocytic removal, which is a normal physiological process during development, but a pathological process in several neurodegenerative diseases and conditions. We discuss the role of complement in the refinement and elimination of synapses in the retina and lateral geniculate nucleus, both during development and in disease states. How complement and aberrant phagocytosis promotes injury to the visual system is discussed primarily in the context of multiple sclerosis, where it has been extensively studied, although the role of complement in visual dysfunction in other diseases such as stroke and traumatic brain injury is also highlighted. Retinal diseases are also covered, with a focus on glaucoma and age-related macular degeneration. Finally, we discuss the potential of complement inhibitory strategies to treat diseases affecting the visual system.