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A Model for Encephalomyosynangiosis Treatment after Middle Cerebral Artery Occlusion-Induced Stroke in Mice

There is no effective treatment available for most patients suffering with ischemic stroke, making development of novel therapeutics imperative. The ...

UConn School of Medicine

The brain's initial innate response to stroke is primarily mediated by microglia, the resident macrophage of the CNS. However, as early as 4 h after stroke, the blood-brain barrier is compromised and monocyte infiltration occurs. The lack of discriminating markers between these two myeloid populations has led many studies to generate conclusions based on the grouping of these two populations. A growing body of evidence now supports the distinct roles played by microglia and monocytes in many disease models.

Functional differences between microglia and monocytes after ischemic stroke.

Low levels of brain-derived neurotrophic factor (BDNF) are linked to delayed neurological recovery, depression, and cognitive impairment following stroke. Supplementation with BDNF reverses these effects. Unfortunately, systemically administered BDNF in its native form has minimal therapeutic value due to its poor blood brain barrier permeability and short serum half-life. In this study, a novel nano-particle polyion complex formulation of BDNF (nano-BDNF) was administered to mice after experimental ischemic stroke.

Nano-particle delivery of brain derived neurotrophic factor after focal cerebral ischemia reduces tissue injury and enhances behavioral recovery.

Acute ischemic injury leads to severe neuronal loss. One of the key mechanisms responsible for this effect is inflammation, which is characterized by the activation of myeloid cells, including resident microglia and infiltrating monocytes/macrophages. P2X4 receptors (P2X4Rs) present on these immune cells modulate the inflammatory response. For example, excessive release of adenosine triphosphate during acute ischemic stroke triggers stimulation of P2X4Rs, leading to myeloid cell activation and proliferation and further exacerbating post-ischemic inflammation. In contrast, during recovery P2X4Rs activation on microglia leads to the release of brain-derived neurotrophic factor (BDNF), which alleviate depression, maintain synaptic plasticity and hasten post-stroke behavioral recovery. Therefore, we hypothesized that deletion of the P2X4R specifically from myeloid cells would have differential effects on acute versus chronic recovery following stroke.

Deletion of the P2X4 receptor is neuroprotective acutely, but induces a depressive phenotype during recovery from ischemic stroke.

Sirtuins (Sirt) are a family of NAD+ dependent histone deacetylase (HDAC) proteins implicated in aging, cell cycle regulation, and metabolism. These proteins are involved in the epigenetic modification of neuromodulatory proteins after strokevia acetylation/deacetylation. The specific role of Sirt3, a mitochondrial sirtuin, in post-stroke injury has been relatively unexplored. Using male Sirt3 knockout (KO) mice and wild-type littermates (WT), we show that Sirt3 KO mice show significant neuroprotection at 3 days after ischemia/reperfusion (I/R) or stroke injury. The deacetylation activity of Sirt3, measured as the amount of reduced acetylated lysine, was increased after stroke. Stroke-induced increases in liver kinase 1 (LKB1) activity were also reduced in KO mice at 3 days after stroke. On further investigation, we found that the levels of Sirt1, another important member of the Sirtuin family, were increased in the brains of Sirt3 KO mice after stroke. To determine the translational relevance of these findings, we then tested the effects of pharmacological inhibition of Sirt3. We found no benefit of Sirt3 inhibition despite clear evidence of deacetylation. Overall, these data suggest that Sirt3 KO mice show neuroprotection by a compensatory rise in Sirt1 rather than the loss of Sirt3 after stroke. Further analysis reveals that the beneficial effects of Sirt1 might be mediated by a decrease in LKB1 activity after stroke. Finally, our data clearly demonstrate the importance of using both pharmacological and genetic methods in pre-clinical stroke studies.

Evaluation of the Neuroprotective Effect of Sirt3 in Experimental Stroke.

Stroke is a leading cause of disability worldwide and hence remains a major medical concern. Besides several pathological features, such as excitotoxicity, peri-infarct depolarization, acidosis, reactive oxygen species generation, apoptosis, and necrosis, dysregulation of the immune system severely affects stroke outcomes. After stroke onset, microglia - the brain-resident macrophage immune cells - and peripheral immune cells affect stoke injury/recovery by releasing pro-inflammatory and/or anti-inflammatory cytokines depending on their microenvironment. These pro- or anti-inflammatory cytokines further affect integrity of the blood brain barrier (BBB) and modulate immune infiltration after stroke. Among peripheral immune cells, bone marrow-derived macrophages (BMDMs) play a critical role in stroke pathology which peaks between three and seven days post-stroke. BMDMs have been extensively studied for their role in exacerbation of stroke injury, however they have rarely been studied for their role in tissue repair. Nonetheless, these reparative roles are gaining attention since recent studies have shown either failure or worsening of long-term post-stroke recovery after blockade of peripheral immune infiltration. These diverse but paradoxical effects of infiltrating monocytes/macrophages encouraged us to summarize the latest findings in neuro-immune and immune-vascular interactions. This review highlights the multifaceted role of BMDMs in stroke onset and resolution, and emphasizes the significance of tapping the potential of these cells to gain better insight into disease progression and therapy.

Role of bone marrow-derived macrophages (BMDMs) in neurovascular interactions during stroke.

Dysregulation of microRNAs (miRNAs) has been tied to several neurological disorders, including ischemic stroke. It has also been established that social environments can modulate miRNA profiles. We have previously shown that post-stroke social isolation (SI) is linked to poor stroke outcomes and that miR-181c-5p emerged as one of few lead miRNAs that was downregulated in both stroke and SI. Therefore, in this study we examined the potential role of miR-181c-5p mimic in reversing the detrimental effects of post-stroke SI. Two to three-month-old C57BL/6 male mice were pair-housed (PH) for at least two weeks. After two weeks, mice underwent stroke survival surgery using middle cerebral artery occlusion (MCAO) and were randomly assigned to one of two housing conditions: stroke isolation (ST-ISO) or stroke pair-housing with a healthy partner (ST-PH). ST-ISO mice were randomized to receive either miR-181c-5p mimic or a scrambled RNA (7 mg/kg i.v./day×drug) control at 24 h and 48 h after stroke. The effects of miR-181c-5p mimic treatment were evaluated at 1, 3, and 7 days after stroke at histological, behavioral, and biochemical levels. Target genes of miR-181c-5p were then analyzed by qPCR using an RT Profiler qPCR Array of pre-coated miR-181c gene targets. Temporal profile expression data suggested that miR-181c-5p was significantly downregulated (p < 0.05 vs ST-PH) up to 7 days after post-stroke SI. MiR-181c-5p mimic treatment significantly increased miR-181c-5p expression in brain tissue and showed partial swift recovery in sensorimotor deficit. Target gene analysis identified downregulation of several calcium signaling-related genes, e.g., Cpne2 and Gria 1 & 2 after miR-181c-5p mimic treatment. In summary, present data suggests that miR-181c-5p is a potential target for post-stroke SI. Data also suggests that genes related to calcium and glutamate signaling might be involved in the beneficial effect of the miR-181c-5p mimic.

Micro RNA 181c-5p: A promising target for post-stroke recovery in socially isolated mice.

Stroke remains a leading cause of disability in the United States. Despite recent advances, interventions to reduce damage and enhance recovery after stroke are lacking. P2X4R, a receptor for adenosine triphosphate (ATP), regulates activation of myeloid immune cells (infiltrating monocytes/macrophages and brain-resident microglia) after stroke injury. However, over-stimulation of P2X4Rs due to excessive ATP release from dying or damaged neuronal cells can contribute to ischemic injury. Therefore, we pharmacologically inhibited P2X4R to limit the over-stimulated myeloid cell immune response and improve both acute and chronic stroke recovery. We subjected 8-12-week-old male and female wild type mice to a 60 min right middle cerebral artery occlusion (MCAo) followed by 3 or 30 days of reperfusion. We performed histological, RNA sequencing, behavioral (sensorimotor, anxiety, and depressive), and biochemical (Evans blue dye extravasation, western blot, quantitative PCR, and flow cytometry) analyses to determine the acute (3 days after MCAo) and chronic (30 days after MCAo) effects of P2X4R antagonist 5-BDBD (1 mg/kg P.O. daily x 3 days post 4 h of MCAo) treatment. 5-BDBD treatment significantly (p < .05) reduced infarct volume, neurological deficit (ND) score, levels of cytokine interleukin-1 beta (IL-1β) and blood brain barrier (BBB) permeability in the 3-day group. Chronically, 5-BDBD treatment also conferred progressive recovery (p < .05) of motor balance and coordination using a rotarod test, as well as reduced anxiety-like behavior over 30 days. Interestingly, depressive-type behavior was not observed in mice treated with 5-BDBD for 3 days. In addition, flow cytometric analysis revealed that 5-BDBD treatment decreased the total number of infiltrated leukocytes, and among those infiltrated leukocytes, pro-inflammatory cells of myeloid origin were specifically reduced. 5-BDBD treatment reduced the cell surface expression of P2X4R in flow cytometry-sorted monocytes and microglia without reducing the total P2X4R level in brain tissue. In summary, acute P2X4R inhibition protects against ischemic injury at both acute and chronic time-points after stroke. Reduced numbers of infiltrating pro-inflammatory myeloid cells, decreased surface P2X4R expression, and reduced BBB disruption are likely its mechanism of neuroprotection and neuro-rehabilitation.

Rajkumar Verma

Department of Neuroscience

UConn School of Medicine

A Model for Encephalomyosynangiosis Treatment after Middle Cerebral Artery Occlusion-Induced Stroke in Mice