Astrocytes in functional recovery following central nervous system injuries.

Astrocytes are increasingly recognised as partaking in complex homeostatic mechanisms critical for regulating neuronal plasticity following central nervous system (CNS) insults. Ischaemic stroke and traumatic brain injury are associated with high rates of disability and mortality. Depending on the context and type of injury, reactive astrocytes respond with diverse morphological, proliferative and functional changes collectively known as astrogliosis, which results in both pathogenic and protective effects. There is a large body of research on the negative consequences of astrogliosis following brain injuries. There is also growing interest in how astrogliosis might in some contexts be protective and help to limit the spread of the injury. However, little is known about how astrocytes contribute to the chronic functional recovery phase following traumatic and ischaemic brain insults. In this review, we explore the protective functions of astrocytes in various aspects of secondary brain injury such as oedema, inflammation and blood-brain barrier dysfunction. We also discuss the current knowledge on astrocyte contribution to tissue regeneration, including angiogenesis, neurogenesis, synaptogenesis, dendrogenesis and axogenesis. Finally, we discuss diverse astrocyte-related factors that, if selectively targeted, could form the basis of astrocyte-targeted therapeutic strategies to better address currently untreatable CNS disorders.

Expression of miR-200c corresponds with increased reactive oxygen species and hypoxia markers after transient focal ischemia in mice.

Embolic stroke results in a necrotic core of cells destined to die, but also a peri-ischemic, watershed penumbral region of potentially salvageable brain tissue. Approaches to effectively differentiate between the ischemic and peri-ischemic zones is critical for novel therapeutic discovery to improve outcomes in survivors of stroke. MicroRNAs are a class of small non-coding RNAs regulating gene translation that have region- and cell-specific expression and responses to ischemia. We have previously reported that global inhibition of cerebral microRNA-200c after experimental stroke in mice is protective, however delineating the post-stroke sub-regional and cell-type specific patterns of post-stroke miR-200c expression are necessary to minimize off-target effects and advance translational application. Here, we detail a novel protocol to visualize regional miR-200c expression after experimental stroke, complexed with visualization of regional ischemia and markers of oxidative stress in an experimental stroke model in mice. In the present study we demonstrate that the fluorescent hypoxia indicator pimonidazole hydrochloride, the reactive-oxygen-species marker 8-hydroxy-deoxyguanosine, neuronal marker MAP2 and NeuN, and the reactive astrocyte marker GFAP can be effectively complexed to determine regional differences in ischemic injury as early as 30 min post-reperfusion after experimental stroke, and can be effectively used to distinguish ischemic core from surrounding penumbral and unaffected regions for targeted therapy. This multi-dimensional post-stroke immunofluorescent imaging protocol enables a greater degree of sub-regional mechanistic investigation, with the ultimate goal of developing more effective post-stroke pharmaceutical therapy.

Reduction of Inflammation by High-Dose Methylprednisolone Does not Attenuate Oxidative Stress in Children Undergoing Bidirectional Glenn Procedure With or Without Aortic Arch or Pulmonary Arterial Repair.

OBJECTIVE: Corticosteroids attenuate an inflammatory reaction in pediatric heart surgery. Inflammation is a source of free oxygen radicals. Children with a cyanotic heart defect are prone to increased radical stress during heart surgery. The authors hypothesized that high-dose methylprednisolone reduces inflammatory reaction and thereby also oxidative stress in infants with a univentricular heart defect undergoing the bidirectional Glenn procedure. DESIGN: A double-blind, placebo-controlled, randomized clinical trial. SETTING: Operating room and pediatric intensive care unit of a university hospital. PARTICIPANTS: The study comprised 29 infants undergoing the bidirectional Glenn procedure with or without aortic arch or pulmonary arterial repair. INTERVENTIONS: After anesthesia induction, the patients received intravenously either 30 mg/kg of methylprednisolone (n = 15) or the same volume of saline as placebo (n = 14). MEASUREMENTS AND MAIN RESULTS: Plasma interleukin-6, interleukin-8, interleukin-10 (biomarkers of inflammation), and 8-hydroxydeoxyguanosine concentrations (a biomarker of oxidative stress) were measured at the following 4 time points: preoperatively, during cardiopulmonary bypass, after protamine administration, and 6 hours postoperatively. The study parameters did not differ between the study groups preoperatively. Methylprednisolone reduced the proinflammatory cytokines interleukin-6 and interleukin-8 and increased the anti-inflammatory cytokine interleukin-10 postoperatively. Despite reduced inflammation, there were no differences in 8-hydroxydeoxyguanosine between the methylprednisolone and placebo groups. CONCLUSIONS: The proinflammatory reaction and increase in free radical stress were not interrelated during congenital heart surgery in cyanotic infants with a univentricular heart defect undergoing the bidirectional Glenn procedure. High-dose methylprednisolone was ineffective in attenuating free radical stress.

Stem Cell-Derived Exosomes Protect Astrocyte Cultures From in vitro Ischemia and Decrease Injury as Post-stroke Intravenous Therapy.

In the present study, we assessed efficacy of exosomes harvested from human and mouse stem cell cultures in protection of mouse primary astrocyte and neuronal cell cultures following in vitro ischemia, and against ischemic stroke in vivo. Cell media was collected from primary mouse neural stem cell (NSC) cultures or from human induced pluripotent stem cell-derived cardiomyocyte (iCM) cultures. Exosomes were extracted and purified by polyethylene glycol complexing and centrifugation, and exosome size and concentration were determined with a NanoSiteTM particle analyzer. Exosomes were applied to primary mouse cortical astrocyte or neuronal cultures prior to, and/or during, combined oxygen-glucose deprivation (OGD) injury. Cell death was assessed via lactate dehydrogenase (LHD) and propidium iodide staining 24 h after injury. NSC-derived exosomes afforded marked protection to astrocytes following OGD. A more modest (but significant) level of protection was observed with human iCM-derived exosomes applied to astrocytes, and with NSC-derived exosomes applied to primary neuronal cultures. In subsequent experiments, NSC-derived exosomes were injected intravenously into adult male mice 2 h after transient (1 h) middle cerebral artery occlusion (MCAO). Gross motor function was assessed 1 day after reperfusion and infarct volume was assessed 4 days after reperfusion. Mice treated post-stroke with intravenous NSC-derived exosomes exhibited significantly reduced infarct volumes. Together, these results suggest that exosomes isolated from mouse NSCs provide neuroprotection against experimental stroke possibly via preservation of astrocyte function. Intravenous NSC-derived exosome treatment may therefore provide a novel clinical adjuvant for stroke in the immediate post-injury period.

Nursing Markedly Protects Postpartum Mice From Stroke: Associated Central and Peripheral Neuroimmune Changes and a Role for Oxytocin.

Recent studies demonstrate significant neuroimmune changes in postpartum females, a period that also carries an increased risk of stroke. Oxytocin, a major hormone upregulated in the brains of nursing mothers, has been shown to both modulate neuroinflammation and protect against stroke. In the present study we assessed whether and how nursing modulates the neuroimmune response and injury after stroke. We observed that postpartum nursing mice were markedly protected from 1 h of transient middle cerebral artery occlusion (MCAO) relative to either non-pregnant/non-postpartum or non-nursing (pups removed) postpartum females. Nursing mice also expressed reduced levels of pro-inflammatory cytokines, had decreased migration of blood leukocytes into the brain following MCAO, and displayed peripheral neuroimmune changes characterized by increased spleen weight and increased fraction of spleen monocytes. Intranasal oxytocin treatment in non-pregnant females in part recapitulated the protective and anti-inflammatory effects associated with nursing. In summary, the results of the present study demonstrate that nursing in the postpartum period provides relative protection against transient ischemic stroke associated with decreased brain leukocytes and increased splenic monocytes. These effects appear to be regulated, at least in part, by oxytocin.

Hippocampal sub-regional differences in the microRNA response to forebrain ischemia.

Transient forebrain ischemia, as occurs with cardiac arrest and resuscitation, results in impaired cognitive function secondary to delayed neuronal cell death in hippocampal cornu ammonis-1 (CA1). Comparatively, hippocampal neurons in the adjacent dentate gyrus (DG) survive, suggesting that elucidating the molecular mechanisms underpinning hippocampal sub-regional differences in ischemic tolerance could be central in the development of novel interventions to improve outcome in survivors of forebrain ischemia. MicroRNAs (miRNAs) are non-coding RNAs that modulate the translation of target genes and have been established as an effective therapeutic target for other models of injury. The objective of the present study was to assess and compare post-injury miRNA profiles between CA1 and DG using a rat model of forebrain ischemia. CA1 and DG sub-regions were dissected from rat hippocampi following 10 min of forebrain ischemia at three time points (3 h, 24 h, and 72 h) and at baseline. Pronounced differences between CA1 and DG were observed for several select miRNAs, including miR-181a-5p, a known regulator of cerebral ischemic injury. We complexed fluorescent in situ hybridization with immunohistochemistry to observe cell-type specific and temporal differences in mir-181a-5p expression between CA1 and DG in response to injury. Using established miRNA-mRNA prediction algorithms, we extended our observations in CA1 miRNA dysregulation to identify key functional pathways as potential modulators of CA1 ischemic vulnerability. In summary, our observations support a central role for miRNAs in selective CA1 ischemic vulnerability and suggest that cell-specific miRNA targeting could be a viable clinical approach to preserve CA1 neurons and improve cognitive outcomes for survivors of transient forebrain ischemia.

Pre-treatment with microRNA-181a Antagomir Prevents Loss of Parvalbumin Expression and Preserves Novel Object Recognition Following Mild Traumatic Brain Injury.

Mild traumatic brain injury (mTBI) can result in permanent impairment in memory and learning and may be a precursor to other neurological sequelae. Clinical treatments to ameliorate the effects of mTBI are lacking. Inhibition of microRNA-181a (miR-181a) is protective in several models of cerebral injury, but its role in mTBI has not been investigated. In the present study, miR-181a-5p antagomir was injected intracerebroventricularly 24 h prior to closed-skull cortical impact in young adult male mice. Paw withdrawal, open field, zero maze, Y maze, object location and novel object recognition tests were performed to assess neurocognitive dysfunction. Brains were assessed immunohistologically for the neuronal marker NeuN, the perineuronal net marker wisteria floribunda lectin (WFA), cFos, and the interneuron marker parvalbumin. Protein quantification was performed with immunoblots for synaptophysin and postsynaptic density 95 (PSD95). Fluorescent in situ hybridization was utilized to localize hippocampal miR-181a expression. MiR-181a antagomir treatment reduced neuronal miR-181a expression after mTBI, restored deficits in novel object recognition and increased hippocampal parvalbumin expression in the dentate gyrus. These changes were associated with decreased dentate gyrus hyperactivity indicated by a relative reduction in PSD95 and cFos expression. These results suggest that miR-181a inhibition may be a therapeutic approach to reduce hippocampal excitotoxicity and prevent cognitive dysfunction following mTBI.

Pharmacological Preconditioning with Diazoxide in the Experimental Hypothermic Circulatory Arrest Model.

BACKGROUND: Hypothermic circulatory arrest includes a remarkable risk for neurological injury. Diazoxide, a mitochondrial adenosine triphosphate-dependent potassium ion (K+ATP) channel opener, is known to have cardioprotective effects. We assessed its efficacy in preventing ischemic injury in a clinically relevant animal model. Methods: Eighteen piglets were randomized into a diazoxide group (n = 9) and a control group (n = 9). Animals underwent 60 minutes of hypothermic circulatory arrest at 18°C. Diazoxide (5 mg/kg + 10 mL NaOH + 40 mL NaCl) was infused during the cooling phase. Metabolic and hemodynamic data were collected throughout the experiment. After 24-hour follow-up, whole brain, heart, and kidney biopsy specimens were collected for analysis. Results: Cerebellar Cytochrome-C and caspase-3 activation was higher in the control group (P = .02 and P = .016, respectively). Antioxidant activity tended to be higher in the diazoxide group (P = .099). Throughout the experiment, the oxygen consumption ratio was higher in the control animals (Pg = .04), as were the lactate levels (Pg = .02). Cardiac function tended to be better in diazoxide-treated animals. Conclusion: Diazoxide might confer neuroprotective effect as implied by the immunohistochemical analysis of the brain. Additionally, the circulatory effects of diazoxide were beneficial, supporting its neuroprotective effect.

Remote Ischemic Preconditioning Attenuates Oxidative Stress during Cardiopulmonary Bypass.

BACKGROUND: Deep hypothermic circulatory arrest (DHCA) is used to overcome the threat of cerebral ischemia during complex surgical operations of the heart and the aortic arch. Remote ischemic preconditioning (RIPC) has been shown to mitigate neurological damage. METHODS: We analyzed blood samples in a consecutive series of 52 piglets that underwent a 60-min period of DHCA with RIPC (the RIPC group) or without (the control group), to reveal whether the protective effect to oxidative stress could be seen by measuring serum 8-hydroxydeoxyguanosine (8-OHdG). The piglets were cannulated and cooled to 18°C using a heart-lung machine, for the DHCA. The piglets were then rewarmed to normothermic temperature. Blood sampling was taken at baseline, after 30 minutes of cooling, 2 hours postoperatively, and 8 hours postoperatively, and analyzed. 8-hydroxydeoxyguanosine (8-OHdG) from blood samples was analyzed by using Enzyme Linked Immunosorbent Assay (ELISA). RESULTS: The serum 8-OHdG concentration was lower in the RIPC group after the cooling phase, 1.84 (1.44-2.17) ng/mL, and at 8 hours after HCA 1.48 (1.39-1.69) ng/mL, when compared with the control group, where the values were 2.14 (1.81-2.56) and 1.84 (1.62-2.44) ng/mL, respectively (P = .025) and (P = .004). CONCLUSION: Remote ischemic preconditioning lowers oxidative stress during cardiopulmonary bypass.

Remote Ischemic Preconditioning Reduces Cerebral Oxidative Stress Following Hypothermic Circulatory Arrest in a Porcine Model.

Remote ischemic precondition has become prominent as one of the most promising methods to mitigate neurological damage following ischemic insult. The purpose of this study was to investigate whether the effects of remote ischemic preconditioning can be seen in the markers of oxidative stress or in redox-regulating enzymes in a porcine model. A total of 12 female piglets were randomly assigned to 2 groups. The study group underwent an intervention of 4 cycles of 5-minute ischemic preconditioning on the right hind leg. All piglets underwent 60-minute hypothermic circulatory arrest. Oxidative stress marker 8-hydroxydeoxyguanosine (8-OHdG) was measured from blood samples with enzyme-linked immunosorbent assay. After 7 days of follow-up, samples from the brain, heart, kidney, and ovary were harvested for histopathologic examination. The immunohistochemical stainings of hypoxia marker hypoxia-inducible factor-1-α, oxidative stress marker 8-OHdG, DNA repair enzyme 8-oxoguanine glycosylase, and antioxidant response regulators nuclear factor erythroid 2-related factor 2 and protein deglycase were analyzed. The level of 8-OHdG referred to baseline was decreased in the sagittal sinus׳ blood samples in the study group after a prolonged deep hypothermic circulatory arrest at 360 minutes after reperfusion. Total histopathologic score was 3.8 (1.8-6.0) in the study group and was 4.4 (2.5-6.5) in the control group (P = 0.72), demonstrating no statistically significant difference in cerebral injury. Our findings demonstrate that the positive effects of remote ischemic preconditioning can be seen in cellular oxidative balance regulators in an animal model after 7 days of preconditioned ischemic insult.

Remote ischemic preconditioning protects the spinal cord against ischemic insult: An experimental study in a porcine model.

OBJECTIVE: Surgical repair of thoracoabdominal aneurysm jeopardizes the vascularization of the spinal cord, and therefore, despite improvement in surgical techniques, still carries the risk of paraplegia. This study aimed to demonstrate the possible protective effects of remote ischemic preconditioning (RIPC) on the preservation of spinal cord function after segmental artery (SA) occlusion. METHODS: Twenty piglets were randomized into the RIPC group (n = 10) and the control group (n = 10). The RIPC group underwent transient left hind limb ischemia before systematic left subclavian artery and SA occlusion at the level of the diaphragm. Motor-evoked potential (MEP) monitoring was performed from the hind limbs. Afterward, the thoracic and lumbar spinal cords were harvested and analyzed. RESULTS: The elevation of the MEP amplitude after RIPC was statistically significant, whereas amplitude was consistently decreased in the control group. Additionally, the onset latency was significantly shorter after RIPC during SA occlusion. The control group reached a 50% decrease of MEP amplitude in the right hind limb sooner than did the experimental group. CONCLUSIONS: Remote ischemic preconditioning preserves spinal cord function after left subclavian artery and SA occlusion, as indicated by the MEP amplitudes.

Safety and biodistribution study of bone marrow-derived mesenchymal stromal cells and mononuclear cells and the impact of the administration route in an intact porcine model.

BACKGROUND AIMS: Bone marrow mononuclear cells (BM-MNCs) and bone marrow-derived mesenchymal stem stromal cells (BM-MSCs) could have therapeutic potential for numerous conditions, including ischemia-related injury. Cells transplanted intravascularly may become entrapped in the lungs, which potentially decreases their therapeutic effect and increases the risk for embolism. METHODS: Twelve pigs were divided into groups of 3 and received (99m)Tc- hydroxymethyl-propylene-amine-oxime-labeled autologous BM-MNCs or allogeneic BM-MSCs by either intravenous (IV) or intra-arterial (IA) transplantation. A whole body scan and single photon emission computed tomography/computed tomography (SPECT/CT) were performed 8 h later, and tissue biopsies were collected for gamma counting. A helical CT scan was also performed on 4 pigs to detect possible pulmonary embolism, 2 after IV BM-MSC injection and 2 after saline injection. RESULTS: The transplantation route had a greater impact on the biodistribution of the BM-MSCs than the BM-MNCs. The BM-MNCs accumulated in the spleen and bones, irrespective of the administration route. The BM-MSCs had relatively higher uptake in the kidneys. The IA transplantation decreased the deposition of BM-MSCs in the lungs and increased uptake in other organs, especially in the liver. Lung atelectases were frequent due to mechanical ventilation and attracted transplanted cells. CT did not reveal any pulmonary embolism. CONCLUSIONS: Both administration routes were found to be safe, but iatrogenic atelectasis might be an issue when cells accumulate in the lungs. The IA administration is effective in avoiding pulmonary entrapment of BM-MSCs. The cell type and administration method both have a major impact on the acute homing.

Leg ischaemia before circulatory arrest alters brain leucocyte count and respiratory chain redox state.

OBJECTIVES: Remote ischaemic preconditioning and its neuroprotective abilities are currently under investigation and the method has shown significant effects in several small and large animal studies. In our previous studies, leucocyte filtration during cardiopulmonary bypass reduced cerebrocortical adherent leucocyte count and mitigated cerebral damage after hypothermic circulatory arrest (HCA) in piglets. This study aimed to obtain and assess direct visual data of leucocyte behaviour in cerebral vessels after hypothermic circulatory arrest following remote ischaemic preconditioning. METHODS: Twelve native stock piglets were randomized into a remote ischaemic preconditioning group (n = 6) and a control group (n = 6). The intervention group underwent hind-leg ischaemia, whereas the control group received a sham-treatment before a 60-min period of hypothermic circulatory arrest. An intravital microscope was used to obtain measurements from the cerebrocortical vessel in vivo. It included three sets of filters: a violet filter to visualize microvascular perfusion and vessel diameter, a green filter for visualization of rhodamine-labelled leucocytes and an ultraviolet filter for reduced nicotinamide adenine dinucleotide (NADH) analysis. The final magnification on the microscope was 400. After the experiment, cerebral and cerebellar biopsies were collected and analysed with transmission electron microscope by a blinded analyst. RESULTS: In the transmission electron microscope analysis, the entire intervention group had normal, unaffected rough endoplasmic reticulum's in their cerebellar tissue, whereas the control group had a mean score of 1.06 (standard deviation 0.41) (P = 0.026). The measured amount of adherent leucocytes was lower in the remote ischaemic preconditioning group. The difference was statistically significant at 5, 15 and 45 min after circulatory arrest. Statistically significant differences were seen also in the recovery phase at 90 and 120 min after reperfusion. Nicotinamide adenine dinucleotide autofluorescence had statistically significant differences at 10 min after cooling and at 120 and 180 min after hypothermic circulatory arrest. CONCLUSIONS: Remote ischaemic preconditioning seems to provide better mitochondrial respiratory chain function as indicated by the higher NADH content. It simultaneously provides a reduction of adherent leucocytes in cerebral vessels after hypothermic circulatory arrest. Additionally, it might provide some degree of cellular organ preservation as implied by the electron microscopy results.

Remote ischemic precondition preserves cerebral oxygen tension during hypothermic circulatory arrest.

OBJECTIVES: Remote ischemic preconditioning (RIPC) is a novel and promising method of mitigating neurological injury. In previous animal studies, RIPC has provided substantial neuroprotective effects. We hypothesized that the promising neuroprotective properties were a consequence of a better oxygen consumption profile during hypothermic circulatory arrest (HCA). DESIGN: Six 7-week-old female pigs were randomly assigned to undergo the 60 minutes of HCA with the right hind leg receiving transient RIPC preoperatively and six animals were assigned to a control group that underwent 60 minutes of HCA without any preconditioning. A combined temperature/oxygen-tension probe was inserted into the parietal cortex of each animal to monitor cerebral oxygen tension during experiments. RESULTS: The RIPC group had significantly higher cerebral oxygen tension readings throughout the HCA. Statistically significant differences were measured from the 20 minute time point onwards in every time point up to the 60 minute time point. CONCLUSIONS: This study shows that RIPC performed before HCA conserves the cerebral oxygen tension during a circulatory arrest. RIPC could possibly prolong the safe operating time during HCA as cerebral oxygen content is preserved throughout circulatory arrest.