Association Between Intraventricular Alteplase Use and Parenchymal Hematoma Volume in Patients With Spontaneous Intracerebral Hemorrhage and Intraventricular Hemorrhage.

Importance: Intraventricular thrombolysis reduces intraventricular hemorrhage (IVH) volume in patients with spontaneous intracerebral hemorrhage (ICH), but it is unclear if a similar association with parenchymal ICH volume exists. Objective: To evaluate the association between intraventricular alteplase use and ICH volume as well as the association between a change in parenchymal ICH volume and long-term functional outcomes. Design, Setting, and Participants: This cohort study was a post hoc exploratory analysis of data from the Clot Lysis: Evaluating Accelerated Resolution of Intraventricular Hemorrhage phase 3 randomized clinical trial with blinded outcome assessments. Between September 1, 2009, and January 31, 2015, patients with ICH and IVH were randomized to receive either intraventricular alteplase or normal saline via an external ventricular drain. Participants with primary IVH were excluded. Data analyses were performed between January 1 and June 30, 2021. Exposure: Randomization to receive intraventricular alteplase. Main Outcomes and Measures: The primary outcome was the change in parenchymal ICH volume between the hematoma stability and end-of-treatment computed tomography scans. Secondary outcomes were a modified Rankin Scale score higher than 3 and mortality, both of which were assessed at 6 months. The association between alteplase and change in parenchymal ICH volume was assessed using multiple linear regression, whereas the associations between change in parenchymal ICH volume and 6-month outcomes were assessed using multiple logistic regression. Prespecified subgroup analyses were performed for baseline IVH volume, admission ICH volume, and ICH location. Results: A total of 454 patients (254 men [55.9%]; mean [SD] age, 59 [11] years) were included in the study. Of these patients, 230 (50.7%) were randomized to receive alteplase and 224 (49.3%) to receive normal saline. The alteplase group had a greater mean (SD) reduction in parenchymal ICH volume compared with the saline group (1.8 [0.2] mL vs 0.4 [0.1] mL; P < .001). In the primary analysis, alteplase use was associated with a change in the parenchymal ICH volume in the unadjusted analysis per 1-mL change (ß, 1.37; 95% CI, 0.92-1.81; P < .001) and in multivariable linear regression analysis that was adjusted for demographic characteristics, stability ICH and IVH volumes, ICH location, and time to first dose of study drug per 1-mL change (ß, 1.20; 95% CI, 0.79-1.62; P < .001). In the secondary analyses, no association was found between change in parenchymal ICH volume and poor outcome (odds ratio [OR], 0.97; 95% CI 0.87-1.10; P = .64) or mortality (OR, 0.97; 95% CI 0.99-1.08; P = .59). Similar results were observed in the subgroup analyses. Conclusions and Relevance: This study found that intraventricular alteplase use in patients with a large IVH was associated with a small reduction in parenchymal ICH volume, but this association did not translate into improved functional outcomes or mortality. Intraventricular thrombolysis should be examined in patients with moderate to large ICH with IVH, especially in a thalamic location.

PPAR-γ activation enhances myelination and neurological recovery in premature rabbits with intraventricular hemorrhage.

Intraventricular hemorrhage (IVH) results in periventricular inflammation, hypomyelination of the white matter, and hydrocephalus in premature infants. No effective therapy exists to prevent these disorders. Peroxisome proliferator activated receptor-γ (PPAR-γ) agonists reduce inflammation, alleviate free radical generation, and enhance microglial phagocytosis, promoting clearance of debris and red blood cells. We hypothesized that activation of PPAR-γ would enhance myelination, reduce hydrocephalus, and promote neurological recovery in newborns with IVH. These hypotheses were tested in a preterm rabbit model of IVH; autopsy brain samples from premature infants with and without IVH were analyzed. We found that IVH augmented PPAR-γ expression in microglia of both preterm human infants and rabbit kits. The treatment with PPAR-γ agonist or PPAR-γ overexpression by adenovirus delivery further elevated PPAR-γ levels in microglia, reduced proinflammatory cytokines, increased microglial phagocytosis, and improved oligodendrocyte progenitor cell (OPC) maturation in kits with IVH. Transcriptomic analyses of OPCs identified previously unrecognized PPAR-γ-induced genes for purinergic signaling, cyclic adenosine monophosphate generation, and antioxidant production, which would reprogram these progenitors toward promoting myelination. RNA-sequencing analyses of microglia revealed PPAR-γ-triggered down-regulation of several proinflammatory genes and transcripts having roles in Parkinson's disease and amyotrophic lateral sclerosis, contributing to neurological recovery in kits with IVH. Accordingly, PPAR-γ activation enhanced myelination and neurological function in kits with IVH. This also enhanced microglial phagocytosis of red blood cells but did not reduce hydrocephalus. Treatment with PPAR-γ agonist might enhance myelination and neurological recovery in premature infants with IVH.

Hematoma Expansion Shift Analysis to Assess Acute Intracerebral Hemorrhage Treatments.

OBJECTIVE: Hematoma expansion (HE) is commonly analyzed as a dichotomous outcome in intracerebral hemorrhage (ICH) trials. In this proof-of-concept study, we propose an HE shift analysis model as a method to improve the evaluation of candidate ICH therapies. METHODS: Using data from the Antihypertensive Treatment of Acute Cerebral Hemorrhage II (ATACH-2) trial, we performed HE shift analysis in response to intensive blood pressure lowering by generating polychotomous strata based on previously established HE definitions, percentile/absolute quartiles of hematoma volume change, and quartiles of 24-hour follow-up hematoma volumes. The relationship between blood pressure treatment and HE shift was explored with proportional odds models. RESULTS: The primary analysis population included 863 patients. In both treatment groups, approximately one-third of patients exhibited no HE. With the use of a trichotomous HE stratification, the highest strata of ≥33% revealed a 5.8% reduction in hematoma growth for those randomized to intensive therapy (adjusted odds ratio [aOR] 0.77, 95% confidence interval [CI] 0.60-0.99). Using percentile quartiles of hematoma volume change, we observed a favorable shift to reduce growth in patients treated with intensive therapy (aOR 0.73, 95% CI 0.57-0.93). Similarly, in a tetrachotomous analysis of 24-hour follow-up hematoma volumes, shifts in the highest stratum (>21.9 mL) were most notable. CONCLUSIONS: Our findings suggest that intensive blood pressure reduction may preferentially mitigate growth in patients at risk of high volume HE. A shift analysis model of HE provides additional insights into the biological effects of a given therapy and may be an additional way to assess hemostatic agents in future studies. TRIAL REGISTRATION INFORMATION: ClinicalTrials.gov Identifier:NCT01176565.

White matter injury in infants with intraventricular haemorrhage: mechanisms and therapies.

Intraventricular haemorrhage (IVH) continues to be a major complication of prematurity that can result in cerebral palsy and cognitive impairment in survivors. No optimal therapy exists to prevent IVH or to treat its consequences. IVH varies in severity and can present as a bleed confined to the germinal matrix, small-to-large IVH or periventricular haemorrhagic infarction. Moderate-to-severe haemorrhage dilates the ventricle and damages the periventricular white matter. This white matter injury results from a constellation of blood-induced pathological reactions, including oxidative stress, glutamate excitotoxicity, inflammation, perturbed signalling pathways and remodelling of the extracellular matrix. Potential therapies for IVH are currently undergoing investigation in preclinical models and evidence from clinical trials suggests that stem cell treatment and/or endoscopic removal of clots from the cerebral ventricles could transform the outcome of infants with IVH. This Review presents an integrated view of new insights into the mechanisms underlying white matter injury in premature infants with IVH and highlights the importance of early detection of disability and immediate intervention in optimizing the outcomes of IVH survivors.

Comparative Analysis of the Patients with Spontaneous Thalamic Hemorrhage with Concurrent Intraventricular Hemorrhage and Those without Intraventricular Hemorrhage.

BACKGROUND: This study aimed to compare the characteristics of patients with spontaneous thalamic hemorrhage (STH) accompanied by intraventricular hemorrhage (IVH) with those of patients without IVH. METHODS: The medical records of consecutive patients with STH admitted to our institute between January 2000 and December 2018 were reviewed retrospectively. The laboratory and radiological results, mortality, and functional recovery were compared between the STH patients with IVH and those without IVH. RESULTS: Among 2,389 patients with spontaneous intracerebral hemorrhage, 233 (9.8%) patients were included in this study. Concurrent IVH was detected in 159 (68.2%) patients with STH, and more frequently in those with body mass index ≥ 25, Glasgow Coma Scale score of 3-8, underlying disease, family history of stoke, posterior/medial/global location of hematoma, ventriculomegaly, large volume of hemorrhage, and midline shift ≥ 5 mm. The 3-month mortality was 25.8% and 8.1% (P = 0.039), the rate of good functional recovery at 6 months was 52.2% and 31.0% (P = 0.040), and incidence of delayed normal pressure hydrocephalus (NPH) at 12 months was 10.8% and 24.5% (P = 0.062) in the STH patients with IVH and those without IVH, respectively. At 12 months, delayed NPH developed in 28 of 47 (59.6%) patients who received external ventricular drainage (EVD)-based treatment, 5 of 45 (11.1%) patients who underwent endoscopic evacuation-based treatment, and 8 of 45 (17.8%) patients who underwent other surgeries. CONCLUSION: Concurrent IVH is strongly associated with mortality in patients with STH. Delayed NPH may develop more frequently in STH patients with IVH who were treated with EVD.

Mechanical injury and blood are drivers of spatial memory deficits after rapid intraventricular hemorrhage.

Aneurysmal intraventricular hemorrhage (IVH) survivors may recover with significant deficits in learning and memory. The goal of this study was to investigate the mechanism of memory decline after intraventricular aneurysm rupture. We developed an aneurysmal IVH rat model by injecting autologous, arterial blood over the period of two minutes into the right lateral ventricle. We also evaluated the effects of a volume-matched artificial cerebrospinal fluid (CSF) control, thrombin and the mode of delivery (pulsed hand injection versus continuous pump infusion). We performed magnetic resonance brain imaging after 1 and 5 weeks to evaluate for hydrocephalus and histological analysis of the dentate gyrus after 6 weeks. Only animals which underwent a whole blood pulsed hand injection had a spatial memory acquisition and retention deficit 5 weeks later. These animals had larger ventricles at 1 and 5 weeks than animals which underwent a continuous pump infusion of whole blood. We did not find a decline in dentate gyrus granule cell neurons or an impairment in dentate gyrus neurogenesis or differentiation 6 weeks after IVH. Rapid injections of blood or volume resulted in microglial activation in the dentate gyrus. In conclusion, our results point to mechanical injury as the predominant mechanism of memory decline after intraventricular aneurysmal rupture. However, volume-matched pulsed injections of artificial CSF did not create a spatial memory deficit at 5 weeks. Therefore, whole blood itself must play a role in the mechanism. Further research is required to evaluate whether the viscosity of blood causes additional mechanical disruption and hydrocephalus through a primary injury mechanism or whether the toxicity of blood causes a secondary injury mechanism that leads to the observed spatial memory deficit after 5 weeks.

Preterm intraventricular hemorrhage in vitro: modeling the cytopathology of the ventricular zone.

BACKGROUND: Severe intraventricular hemorrhage (IVH) is one of the most devastating neurological complications in preterm infants, with the majority suffering long-term neurological morbidity and up to 50% developing post-hemorrhagic hydrocephalus (PHH). Despite the importance of this disease, its cytopathological mechanisms are not well known. An in vitro model of IVH is required to investigate the effects of blood and its components on the developing ventricular zone (VZ) and its stem cell niche. To address this need, we developed a protocol from our accepted in vitro model to mimic the cytopathological conditions of IVH in the preterm infant. METHODS: Maturing neuroepithelial cells from the VZ were harvested from the entire lateral ventricles of wild type C57BL/6 mice at 1-4 days of age and expanded in proliferation media for 3-5 days. At confluence, cells were re-plated onto 24-well plates in differentiation media to generate ependymal cells (EC). At approximately 3-5 days, which corresponded to the onset of EC differentiation based on the appearance of multiciliated cells, phosphate-buffered saline for controls or syngeneic whole blood for IVH was added to the EC surface. The cells were examined for the expression of EC markers of differentiation and maturation to qualitatively and quantitatively assess the effect of blood exposure on VZ transition from neuroepithelial cells to EC. DISCUSSION: This protocol will allow investigators to test cytopathological mechanisms contributing to the pathology of IVH with high temporal resolution and query the impact of injury to the maturation of the VZ. This technique recapitulates features of normal maturation of the VZ in vitro, offering the capacity to investigate the developmental features of VZ biogenesis.

Retrospective study of short-term complications associated with early morphine use in intubated premature infants.

Relieving neonatal pain is essential for the management of premature infants. Morphine is the most frequently used analgesic in neonatal intensive care. Here we report the relationship between early morphine infusion and the composite outcome of intraventricular hemorrhage and/or death in intubated premature infants. Infants (gestational age ≤ 32 weeks and birth weight < 1,500 g) intubated on admission were retrospectively evaluated in a large tertiary neonatal intensive care unit. Modified log-Poisson regression with robust variance estimator and Cox regression was applied to adjust the relative risk for infants' outcomes. Of 420 premature infants, 230 (54.7%) received continuous morphine infusion in the first 72 h. Of these, 153 were < 28 gestational weeks; of the 190 patients who did not receive morphine, 63 were < 28 gestational weeks. The analysis revealed that infants < 28 gestational weeks who received morphine were significantly associated with an increased risk for IVH and/or death [adjusted relative risk (aRR) 1.37, 95% confidence interval (CI) 1.1-1.71)], and mortality (aRR 1.83, 95% CI 1.17-2.89). Moreover, in infants < 28 gestational weeks, survival was low in those infants who were exposed to morphine infusion in the first 72 h (hazard ratio 2.11; 95% CI 1.19-3.73). Early morphine infusion is associated with an increased risk for IVH and/or death; however, further studies are required to verify our findings.

The slice score: A novel scale measuring intraventricular hemorrhage severity and predicting poor outcome following intracerebral hemorrhage.

OBJECTIVES: To quantify extent of intraventricular hemorrhage (IVH) following intracerebral hemorrhage (ICH) with a novel, simple IVH severity score, and to explore and compare its performance in predicting worse outcomes. PATIENTS AND METHODS: A new scoring system for IVH severity was proposed and termed Slice score. The Slice score features non-septum pellucidum section, internal capsule section, third ventricle occipital horn section, three standardized scans for scoring the lateral ventricles. 652 scans from 326 subjects were retrospectively analyzed. The correlations between measured IVH volume and Slice score, original Graeb, LeRoux, and IVH score (IVHS) were compared. The association between these scores and clinical outcomes were evaluated using logistic regression. We then identified clinical thresholds of Slice score by balancing the probability of prediction and accuracy. Primary outcome was defined as 90-day poor outcome (modified Rankin Scale score ≥ 4) and secondary outcome was 90-day mortality. RESULTS: Of 326 ICH patients, 122 (37.4%) had poor outcome and 59 (18.1%) died at 3 months. The Slice score showed the highest correlation with measured IVH volume (R = 0.73, R2 = 0.54, p < 0.001). The observed area under the curve were similar among the Slice, original Graeb, LeRoux score, and IVH score for poor outcome (0.633, 0.633, 0.632, 0.634, respectively), and for mortality (0.660, 0.660, 0.660, 0.656, respectively). All IVH scales were independently associated with 90-day poor outcome and mortality with close odds ratio in adjusted models (all odds ratio > 1.07, all p < 0.05). Multivariable Analyses of categorized Slice score revealed optimal thresholds of 6 and 12 for primary and secondary outcomes (odds ratio 4.20, 95% confidence interval 1.82-10.02, p = 0.001; odds ratio 5.41, 95% confidence interval 1.66-17.43, p = 0.005, respectively). CONCLUSIONS: The Slice score correlated highly with the IVH volume, was a reliable volumetric scale for measuring IVH severity, and could be an easy-to-use tool for predicting 90-day poor outcome and mortality in ICH.

Placental Pathology, Cerebral Blood Flow, and Intraventricular Hemorrhage in Preterm Infants: Is There a Link?

BACKGROUND: There is growing evidence to support an association between placental inflammation and neurological sequelae of preterm infants. The goal of this study is to evaluate the relationship between placental pathology, post-natal Doppler cerebral resistive indices (RI's), and intraventricular hemorrhage (IVH) in premature infants. METHODS: In a retrospective cohort study, preterm infants born between 23 0/7 and 32 6/7 weeks' gestation at Parkland Hospital were examined with placental pathology and serial ultrasound Doppler to evaluate for the primary outcome of IVH and death. RESULTS: A total of 255 infants were included, and 166 (65%) had at least one significant placental pathology, most commonly chorioamnionitis. Infants with placental pathologies were significantly more likely to have mothers with clinical chorioamnionitis and to have lower gestational ages. There was no observed association between placental pathology and IVH or death. Secondary analysis demonstrated that resistive indices obtained from the first and second head ultrasounds were not different in infants with IVH. CONCLUSION: In this study, we observed a high rate of placental pathologies but no alterations in cerebral indices on ultrasound, or differences in rates of IVH or death. Additional studies are necessary to delineate the relationship between placental pathology, white matter brain injury, and outcomes.

Successful use of an artificial placenta-based life support system to treat extremely preterm ovine fetuses compromised by intrauterine inflammation.

BACKGROUND: Ex vivo uterine environment therapy is an experimental intensive care strategy for extremely preterm infants born between 21 and 24 weeks of gestation. Gas exchange is performed by membranous oxygenators connected by catheters to the umbilical vessels. The fetus is submerged in a bath of synthetic amniotic fluid. The lungs remain fluid filled, and pulmonary respiration does not occur. Intrauterine inflammation is strongly associated with extremely preterm birth and fetal injury. At present, there are no data that we are aware of to show that artificial placenta-based systems can be used to support extremely preterm fetuses compromised by exposure to intrauterine inflammation. OBJECTIVE: To evaluate the ability of our ex vivo uterine environment therapy platform to support extremely preterm ovine fetuses (95-day gestational age; approximately equivalent to 24 weeks of human gestation) exposed to intrauterine inflammation for a period of 120 hours, the following primary endpoints were chosen: (1) maintenance of key physiological variables within normal ranges, (2) absence of infection and inflammation, (3) absence of brain injury, and (4) gross fetal growth and cardiovascular function matching that of age-matched in utero controls. STUDY DESIGN: Ten ewes with singleton pregnancies were each given a single intraamniotic injection of 10-mg Escherichia coli lipopolysaccharides under ultrasound guidance 48 hours before undergoing surgical delivery for adaptation to ex vivo uterine environment therapy at 95-day gestation (term=150 days). Fetuses were adapted to ex vivo uterine environment therapy and maintained for 120 hours with constant monitoring of key vital parameters (ex vivo uterine environment group) before being killed at 100-day equivalent gestational age. Umbilical artery blood samples were regularly collected to assess blood gas data, differential counts, biochemical parameters, inflammatory markers, and microbial load to exclude infection. Ultrasound was conducted at 48 hours after intraamniotic lipopolysaccharides (before surgery) to confirm fetal viability and at the conclusion of the experiments (before euthanasia) to evaluate cardiac function. Brain injury was evaluated by gross anatomic and histopathologic investigations. Eight singleton pregnant control animals were similarly exposed to intraamniotic lipopolysaccharides at 93-day gestation and were killed at 100-day gestation to allow comparative postmortem analyses (control group). Biobanked samples from age-matched saline-treated animals served as an additional comparison group. Successful instillation of lipopolysaccharides into the amniotic fluid exposure was confirmed by amniotic fluid analysis at the time of administration and by analyzing cytokine levels in fetal plasma and amniotic fluid. Data were tested for mean differences using analysis of variance. RESULTS: Six of 8 lipopolysaccharide control group (75%) and 8 of 10 ex vivo uterine environment group fetuses (80%) successfully completed their protocols. Six of 8 ex vivo uterine environment group fetuses required dexamethasone phosphate treatment to manage profound refractory hypotension. Weight and crown-rump length were reduced in ex vivo uterine environment group fetuses at euthanasia than those in lipopolysaccharide control group fetuses (P<.05). There were no biologically significant differences in cardiac ultrasound measurement, differential leukocyte counts (P>.05), plasma tumor necrosis factor α, monocyte chemoattractant protein-1 concentrations (P>.05), or liver function tests between groups. Daily blood cultures were negative for aerobic and anaerobic growth in all ex vivo uterine environment group animals. No cases of intraventricular hemorrhage were observed. White matter injury was identified in 3 of 6 lipopolysaccharide control group fetuses and 3 of 8 vivo uterine environment group fetuses. CONCLUSION: We report the use of an artificial placenta-based system to support extremely preterm lambs compromised by exposure to intrauterine inflammation. Our data highlight key challenges (refractory hypotension, growth restriction, and white matter injury) to be overcome in the development and use of artificial placenta technology for extremely preterm infants. As such challenges seem largely absent from studies based on healthy pregnancies, additional experiments of this nature using clinically relevant model systems are essential for further development of this technology and its eventual clinical application.

Hematoma enlargement characteristics in deep versus lobar intracerebral hemorrhage.

OBJECTIVE: Hematoma enlargement (HE) is associated with clinical outcomes after supratentorial intracerebral hemorrhage (ICH). This study evaluates whether HE characteristics and association with functional outcome differ in deep versus lobar ICH. METHODS: Pooled analysis of individual patient data between January 2006 and December 2015 from a German-wide cohort study (RETRACE, I + II) investigating ICH related to oral anticoagulants (OAC) at 22 participating centers, and from one single-center registry (UKER-ICH) investigating non-OAC-ICH patients. Altogether, 1954 supratentorial ICH patients were eligible for outcome analyses, which were separately conducted or controlled for OAC, that is, vitamin-K-antagonists (VKA, n = 1186) and non-vitamin-K-antagonist-oral-anticoagulants (NOAC, n = 107). Confounding was addressed using propensity score matching, cox regression modeling and multivariate modeling. Main outcomes were occurrence, extent, and timing of HE (>33%/>6 mL) and its association with 3-month functional outcome. RESULTS: Occurrence of HE was not different after deep versus lobar ICH in patients with non-OAC-ICH (39/356 [11.0%] vs. 36/305 [11.8%], P = 0.73), VKA-ICH (249/681 [36.6%] vs. 183/505 [36.2%], P = 0.91), and NOAC-ICH (21/69 [30.4%] vs. 12/38 [31.6%], P = 0.90). HE extent did not differ after non-OAC-ICH (deep:+59% [40-122] vs. lobar:+74% [37-124], P = 0.65), but both patients with VKA-ICH and NOAC-ICH showed greater HE extent after deep ICH [VKA-ICH, deep: +94% [54-199] vs. lobar: +56% [35-116], P < 0.001; NOAC-ICH, deep: +74% [56-123] vs. lobar: +40% [21-49], P = 0.001). Deep compared to lobar ICH patients had higher HE hazard during first 13.5 h after onset (Hazard ratio [HR]: 1.85 [1.03-3.31], P = 0.04), followed by lower hazard (13.5-26.5 h, HR: 0.46 [0.23-0.89], P = 0.02), and equal hazard thereafter (HR: 0.96 [0.56-1.65], P = 0.89). Odds ratio for unfavorable outcome was higher after HE in deep (4.31 [2.71-6.86], P < 0.001) versus lobar ICH (2.82 [1.71-4.66], P < 0.001), and only significant after small-medium (1st volume-quarter, deep: 3.09 [1.52-6.29], P < 0.01; lobar: 3.86 [1.35-11.04], P = 0.01) as opposed to large-sized ICH (4th volume-quarter, deep: 1.09 [0.13-9.20], P = 0.94; lobar: 2.24 [0.72-7.04], P = 0.17). INTERPRETATION: HE occurrence does not differ among deep and lobar ICH. However, compared to lobar ICH, HE after deep ICH is of greater extent in OAC-ICH, occurs earlier and may be of greater clinical relevance. Overall, clinical significance is more apparent after small-medium compared to large-sized bleedings.

Effects of aging on hydrocephalus after intraventricular hemorrhage.

BACKGROUND: Hydrocephalus is a common and major complication that affects outcome after intraventricular hemorrhage (IVH). While aging impacts the occurrence of hydrocephalus in patients with IVH this and the underlying mechanisms have received little attention. The present investigation, therefore, studied the impact of aging on hydrocephalus after IVH in a rat model. METHODS: Young and aged (3 and 18 months old, respectively) male Fischer 344 rats had an intraventricular injection of 200 µl autologous blood or saline. Ventricular volume was estimated using magnetic resonance imaging (MRI), while ventricular wall damage, heme oxygenase-1 (HO-1) and epiplexus cell activation were quantified by histological staining and Western blot. Additionally, the impact of intraventricular iron injection was examined in young and aged rats. RESULTS: Intraventricular injection of autologous blood induced hydrocephalus in both young and aged rats but ventricular volumes were larger in aged rats compared to young rats from day 3 to day 14 followed IVH. In addition, ventricular wall damage and periventricular HO-1 upregulation were greater in aged versus young rats on day 1 after IVH. Aged rats also had more choroid plexus epiplexus cells on day 14 after IVH. Additionally, organized hematomas were observed in 23% (3/13) of aged rats but not in young rats after IVH. Organized hematomas in aged rats showed larger T2* lesions on MRI compared to rats with non-organized hematomas. Similar to the effects of IVH, intraventricular injection of iron resulted in more epiplexus cells activation and more severe hydrocephalus in aged compared to young rats. CONCLUSIONS: IVH causes more severe hydrocephalus in aged compared to young rats. Enhanced ventricular wall damage, epiplexus cell activation and iron overload may contribute to this aggravated hydrocephalus development in aged animals.

Hemoglobin oxidation generates globin-derived peptides in atherosclerotic lesions and intraventricular hemorrhage of the brain, provoking endothelial dysfunction.

The lysis of red blood cells was shown to occur in human ruptured atherosclerotic lesions and intraventricular hemorrhage (IVH) of the brain. Liberated cell-free hemoglobin was found to undergo oxidation in both pathologies. We hypothesize that hemoglobin-derived peptides are generated during hemoglobin oxidation both in complicated atherosclerotic lesions and IVH of the brain, triggering endothelial cell dysfunction. Oxidized hemoglobin and its products were followed with spectrophotometry, LC-MS/MS analysis and detection of the cross-linking of globin chains in complicated atherosclerotic lesions of the human carotid artery and the hemorrhaged cerebrospinal liquid of preterm infants. The vascular pathophysiologic role of oxidized hemoglobin and the resultant peptides was assessed by measuring endothelial integrity, the activation of endothelial cells and the induction of proinflammatory genes. Peptide fragments of hemoglobin (VNVDEVGGEALGRLLVVYPWTQR, LLVVYPWTQR, MFLSFPTTK, VGAHAGEYGAELERMFLSFPTTK, and FLASVSTVLTSKYR) were identified in ruptured atherosclerotic lesions and in IVH of the human brain. Fragments resulting from the oxidation of hemoglobin were accompanied by the accumulation of ferryl hemoglobin. Similar to complicated atherosclerotic lesions of the human carotid artery, a high level of oxidized and cross-linked hemoglobin was observed in the cerebrospinal fluid after IVH. Haptoglobin inhibited hemoglobin fragmentation provoked by peroxide. The resultant peptides failed to bind haptoglobin or albumin. Peptides derived from hemoglobin oxidation and ferryl hemoglobin induced intercellular gap formation, decreased junctional resistance in the endothelium, and enhanced monocyte adhesion to endothelial cells. Enhanced expression of TNF and the activation of NLRP3 and CASP1 followed by the increased generation of IL-1ß and nuclear translocation of the NF-κß transcription factor occurred in response to hemoglobin-derived peptides, and ferryl hemoglobin in endothelium was upregulated in both pathologies. We conclude that the oxidation of hemoglobin in complicated atherosclerotic lesions and intraventricular hemorrhage of the brain generates peptide fragments and ferryl hemoglobin with the potential to trigger endothelial cell dysfunction.

MicroRNA-152 attenuates neuroinflammation in intracerebral hemorrhage by inhibiting thioredoxin interacting protein (TXNIP)-mediated NLRP3 inflammasome activation.

Neuroinflammation significantly contributes to brain injury and neurological deterioration following intracerebral hemorrhage (ICH). MicroRNA-152(miR-152) was reported to be downregulated in ICH patients and to possess anti-inflammatory properties in other diseases. In this study, we aimed to explore the role of miR-152 in ICH, and the underlying mechanisms, using a collagenase-induced rat ICH model and hemin-exposure as a cell model. We first confirmed that miR-152 was consistently downregulated in both models. Overexpression of miR-152 in microglial BV2 cells reduced hemin-induced inflammatory response and reactive oxygen species (ROS) generation, thus protecting co-cultured neuronal HT22 cells. Moreover, overexpression of miR-152 by intracerebroventricular lentivirus injection in ICH rats significantly alleviated neurodecifits, brain edema, and hematoma. These changes were associated with a marked reduction in ICH-induced neuronal death, as detected by co-staining of NeuN and TUNEL, and ICH-induced neuroinflammation, as revealed by inflammatory cytokine levels as well as by the number of Iba1 positive-stained cells in the perihematomal region. Mechanistically, miR-152 significantly inhibited ICH-induced TXNIP expression, and its overexpression blocked the interaction between TXNIP and NOD-like receptor pyrin domain containing 3(NLRP3), thus inhibiting NLRP3-driven inflammasome activation to attenuate neuroinflammation in vivo and in vitro. Moreover, the results of si-TXNIP transfection further confirmed that TXNIP inhibition was involved in the reduction of NLRP3 inflammasome activation by the overexpression of miR-152. Collectively, the present study demonstrates that miR-152 confers protection against ICH-induced neuroinflammation and brain injury by inhibiting TXNIP-mediated NLRP3 inflammasome activation, indicating a potential strategy for ICH treatment.

Modified behavioural tests to detect white matter injury- induced motor deficits after intracerebral haemorrhage in mice.

Motor function deficit induced by white matter injury (WMI) is one of the most severe complications of intracerebral haemorrhage (ICH). The degree of WMI is closely related to the prognosis of patients after ICH. However, the current behavioural assessment of motor function used in the ICH mouse model is mainly based on that for ischaemic stroke and lacks the behavioural methods that accurately respond to WMI. Here, a series of easy-to-implement behavioural tests were performed to detect motor deficits in mice after ICH. The results showed that the grip strength test and the modified pole test not only can better distinguish the degree of motor dysfunction between different volumes of blood ICH models than the Basso Mouse Scale and the beam walking test but can also accurately reflect the severity of WMI characterized by demyelination, axonal swelling and the latency of motor-evoked potential delay induced by ICH. In addition, after ICH, the results of grip tests and modified pole tests, rather than the Basso Mouse Scale and the beam walking test, were worse than those observed after intraventricular haemorrhage (IVH), which was used as a model of brain haemorrhage in non-white matter areas. These results indicate that the grip strength test and the modified pole test have advantages in detecting the degree of motor deficit induced by white matter injury after ICH in mice.

Brain oxidative damage in murine models of neonatal hypoxia/ischemia and reoxygenation.

The brain is one of the main organs affected by hypoxia and reoxygenation in the neonatal period and one of the most vulnerable to oxidative stress. Hypoxia/ischemia and reoxygenation leads to impairment of neurogenesis, disruption of cortical migration, mitochondrial damage and neuroinflammation. The extent of the injury depends on the clinical manifestation in the affected regions. Preterm newborns are highly vulnerable, and they exhibit severe clinical manifestations such as intraventricular hemorrhage (IVH), retinopathy of prematurity (ROP) and diffuse white matter injury (DWMI) among others. In the neonatal period, the accumulation of high levels of reactive oxygen species exacerbated by the immature antioxidant defense systems in represents cellular threats that, if they exceed or bypass physiological counteracting mechanisms, are responsible of significant neuronal damage. Several experimental models in mice mimic the consequences of perinatal asphyxia and the use of oxygen in the reanimation process that produce brain injury. The aim of this review is to highlight brain damage associated with oxidative stress in different murine models of hypoxia/ischemia and reoxygenation.

The heme and radical scavenger α1-microglobulin (A1M) confers early protection of the immature brain following preterm intraventricular hemorrhage.

BACKGROUND: Germinal matrix intraventricular hemorrhage (GM-IVH) is associated with cerebro-cerebellar damage in very preterm infants, leading to neurodevelopmental impairment. Penetration, from the intraventricular space, of extravasated red blood cells and extracellular hemoglobin (Hb), to the periventricular parenchyma and the cerebellum has been shown to be causal in the development of brain injury following GM-IVH. Furthermore, the damage has been described to be associated with the cytotoxic nature of extracellular Hb-metabolites. To date, there is no therapy available to prevent infants from developing either hydrocephalus or serious neurological disability. Mechanisms previously described to cause brain damage following GM-IVH, i.e., oxidative stress and Hb-metabolite toxicity, suggest that the free radical and heme scavenger α1-microglobulin (A1M) may constitute a potential neuroprotective intervention. METHODS: Using a preterm rabbit pup model of IVH, where IVH was induced shortly after birth in pups delivered by cesarean section at E29 (3 days prior to term), we investigated the brain distribution of recombinant A1M (rA1M) following intracerebroventricular (i.c.v.) administration at 24 h post-IVH induction. Further, short-term functional protection of i.c.v.-administered human A1M (hA1M) following IVH in the preterm rabbit pup model was evaluated. RESULTS: Following i.c.v. administration, rA1M was distributed in periventricular white matter regions, throughout the fore- and midbrain and extending to the cerebellum. The regional distribution of rA1M was accompanied by a high co-existence of positive staining for extracellular Hb. Administration of i.c.v.-injected hA1M was associated with decreased structural tissue and mitochondrial damage and with reduced mRNA expression for proinflammatory and inflammatory signaling-related genes induced by IVH in periventricular brain tissue. CONCLUSIONS: The results of this study indicate that rA1M/hA1M is a potential candidate for neuroprotective treatment following preterm IVH.

GSK3ß Inhibition Restores Impaired Neurogenesis in Preterm Neonates With Intraventricular Hemorrhage.

Intraventricular hemorrhage (IVH) is a common complication of prematurity in infants born at 23-28 weeks of gestation. Survivors exhibit impaired growth of the cerebral cortex and neurodevelopmental sequeale, but the underlying mechanism(s) are obscure. Previously, we have shown that neocortical neurogenesis continues until at least 28 gestational weeks. This renders the prematurely born infants vulnerable to impaired neurogenesis. Here, we hypothesized that neurogenesis is impaired by IVH, and that signaling through GSK3ß, a critical intracellular kinase regulated by Wnt and other pathways, mediates this effect. These hypotheses were tested observationally in autopsy specimens from premature infants, and experimentally in a premature rabbit IVH model. Significantly, in premature infants with IVH, the number of neurogenic cortical progenitor cells was reduced compared with infants without IVH, indicating acutely decreased neurogenesis. This finding was corroborated in the rabbit IVH model, which further demonstrated reduction of upper layer cortical neurons after longer survival. Both the acute reduction of neurogenic progenitors, and the subsequent decrease of upper layer neurons, were rescued by treatment with AR-A014418, a specific inhibitor of GSK3ß. Together, these results indicate that IVH impairs late stages of cortical neurogenesis, and suggest that treatment with GSK3ß inhibitors may enhance neurodevelopment in premature infants with IVH.