Impaired neural differentiation and glymphatic CSF flow in the Ccdc39 rat model of neonatal hydrocephalus: genetic interaction with L1cam.

Neonatal hydrocephalus affects about one child per 1000 births and is a major congenital brain abnormality. We previously discovered a gene mutation within the coiled-coil domain-containing 39 (Ccdc39) gene, which causes the progressive hydrocephalus (prh) phenotype in mice due to lack of ependymal-cilia-mediated cerebrospinal fluid (CSF) flow. In this study, we used CRISPR/Cas9 to introduce the Ccdc39 gene mutation into rats, which are more suitable for imaging and surgical experiments. The Ccdc39prh/prh mutants exhibited mild ventriculomegaly at postnatal day (P)5 that progressed into severe hydrocephalus by P11 (P<0.001). After P11, macrophage and neutrophil invasion along with subarachnoid hemorrhage were observed in mutant brains showing reduced neurofilament density, hypomyelination and increased cell death signals compared with wild-type brains. Significantly more macrophages entered the brain parenchyma at P5 before hemorrhaging was noted and increased expression of a pro-inflammatory factor (monocyte chemoattractant protein-1) was found in the cortical neural and endothelial cells in the mutant brains at P11. Glymphatic-mediated CSF circulation was progressively impaired along the middle cerebral artery from P11 as mutants developed severe hydrocephalus (P<0.001). In addition, Ccdc39prh/prh mutants with L1 cell adhesion molecule (L1cam) gene mutation, which causes X-linked human congenital hydrocephalus, showed an accelerated early hydrocephalus phenotype (P<0.05-0.01). Our findings in Ccdc39prh/prh mutant rats demonstrate a possible causal role of neuroinflammation in neonatal hydrocephalus development, which involves impaired cortical development and glymphatic CSF flow. Improved understanding of inflammatory responses and the glymphatic system in neonatal hydrocephalus could lead to new therapeutic strategies for this condition.This article has an associated First Person interview with the joint first authors of the paper.

Magnetic resonance imaging of disease progression and resolution in a transgenic mouse model of pulmonary fibrosis.

Pulmonary fibrosis contributes to morbidity and mortality in a range of diseases, and there are no approved therapies for reversing its progression. To understand the mechanisms underlying pulmonary fibrosis and assess potential therapies, mouse models are central to basic and translational research. Unfortunately, metrics commonly used to assess murine pulmonary fibrosis require animals to be grouped and euthanized, increasing experimental difficulty and cost. We examined the ability of magnetic resonance imaging (MRI) to noninvasively assess lung fibrosis progression and resolution in a doxycycline (Dox) regulatable, transgenic mouse model that overexpresses transforming growth factor-α (TGF-α) under control of a lung-epithelial-specific promoter. During 7 wk of Dox treatment, fibrotic lesions were readily observed as high-signal tissue. Mean weighted signal and percent signal volume were found to be the most robust MRI-derived measures of fibrosis, and these metrics correlated significantly with pleural thickness, histology scores, and hydroxyproline content (R = 0.75-0.89). When applied longitudinally, percent high signal volume increased by 1.5% wk-1 (P < 0.001) and mean weighted signal increased at a rate of 0.0065 wk-1 (P = 0.0062). Following Dox treatment, lesions partially resolved, with percent high signal volume decreasing by -3.2% wk-1 (P = 0.0034) and weighted mean signal decreasing at -0.015 wk-1 (P = 0.0028). Additionally, longitudinal MRI revealed dynamic remodeling in a subset of lesions, a previously unobserved behavior in this model. These results demonstrate MRI can noninvasively assess experimental lung fibrosis progression and resolution and provide unique insights into its pathobiology.

Blocking lymphocyte trafficking with FTY720 prevents inflammation-sensitized hypoxic-ischemic brain injury in newborns.

Intrauterine infection (chorioamnionitis) aggravates neonatal hypoxic-ischemic (HI) brain injury, but the mechanisms linking systemic inflammation to the CNS damage remain uncertain. Here we report evidence for brain influx of T-helper 17 (TH17)-like lymphocytes to coordinate neuroinflammatory responses in lipopolysaccharide (LPS)-sensitized HI injury in neonates. We found that both infants with histological chorioamnionitis and rat pups challenged by LPS/HI have elevated expression of the interleukin-23 (IL-23) receptor, a marker of early TH17 lymphocytes, in the peripheral blood mononuclear cells. Post-LPS/HI administration of FTY720 (fingolimod), a sphingosine-1-phosphate receptor agonist that blocks lymphocyte trafficking, mitigated the influx of leukocytes through the choroid plexus and acute induction of nuclear factor-κB signaling in the brain. Subsequently, the FTY720 treatment led to attenuated blood-brain barrier damage, fewer cluster of differentiation 4-positive, IL-17A-positive T-cells in the brain, less proinflammatory cytokine, and better preservation of growth and white matter functions. The FTY720 treatment also provided dose-dependent reduction of brain atrophy, rescuing >90% of LPS/HI-induced brain tissue loss. Interestingly, FTY720 neither opposed pure-HI brain injury nor directly inhibited microglia in both in vivo and in vitro models, highlighting its unique mechanism against inflammation-sensitized HI injury. Together, these results suggest that the dual hit of systemic inflammation and neonatal HI injury triggers early onset of the TH17/IL-17-mediated immunity, which causes severe brain destruction but responds remarkably to the therapeutic blockade of lymphocyte trafficking.

Synergy of combined tPA-edaravone therapy in experimental thrombotic stroke.

Edaravone, a potent antioxidant, may improve thrombolytic therapy because it benefits ischemic stroke patients on its own and mitigates adverse effects of tissue plasminogen activator (tPA) in preclinical models. However, whether the combined tPA-edaravone therapy is more effective in reducing infarct size than singular treatment is uncertain. Here we investigated this issue using a transient hypoxia-ischemia (tHI)-induced thrombotic stroke model, in which adult C57BL/6 mice were subjected to reversible ligation of the unilateral common carotid artery plus inhalation of 7.5% oxygen for 30 min. While unilateral occlusion of the common carotid artery suppressed cerebral blood flow transiently, the addition of hypoxia triggered reperfusion deficits, endogenous thrombosis, and attenuated tPA activity, leading up to infarction. We compared the outcomes of vehicle-controls, edaravone treatment, tPA treatment at 0.5, 1, or 4 h post-tHI, and combined tPA-edaravone therapies with mortality rate and infarct size as the primary end-points. The best treatment was further compared with vehicle-controls in behavioral, biochemical, and diffusion tensor imaging (DTI) analyses. We found that application of tPA at 0.5 or 1 h--but not at 4 h post-tHI--significantly decreased infarct size and showed synergistic (p<0.05) or additive benefits with the adjuvant edaravone treatment, respectively. The acute tPA-edaravone treatment conferred >50% reduction of mortality, ∼ 80% decline in infarct size, and strong white-matter protection. It also improved vascular reperfusion and decreased oxidative stress, inflammatory cytokines, and matrix metalloproteinase activities. In conclusion, edaravone synergizes with acute tPA treatment in experimental thrombotic stroke, suggesting that clinical application of the combined tPA-edaravone therapy merits investigation.

Intranasal delivery of cell-penetrating anti-NF-κB peptides (Tat-NBD) alleviates infection-sensitized hypoxic-ischemic brain injury.

Perinatal infection aggravates neonatal hypoxic-ischemic (HI) brain injury and may interfere with therapeutic hypothermia. While the NF-κB signaling pathway has been implicated in microglia activation in infection-sensitized HI, the current therapeutic strategies rely on systemic intervention, which could impair neonatal immunity and increase the risk of severe infection. To devise a brain-targeted anti-NF-κB strategy, we examined the effects of intranasal delivery of tat-NBD peptides in two animal models of neonatal infection-sensitized HI. Kinetic experiments showed that tat-NBD peptides entered the olfactory bulbs rapidly (10-30 min) and peaked in the cerebral cortex around 60 min after intranasal application in P7 rats. Further, intranasal delivery of 1.4 mg/kg tat-NBD, which is only 7% of the intravenous dose in past studies, markedly attenuated NF-κB signaling, microglia activation, and brain damage triggered by HI with 4 or 72 h pre-exposure to the bacterial endotoxin lipopolysaccharide (LPS). In contrast, intranasal delivery of mutant tat-NBD peptides or systemic application of minocycline failed to block LPS-sensitized HI injury. Yet, intranasal delivery of up to 5.6 mg/kg tat-NBD peptides immediately after pure-HI insult showed little protection, likely due to its rapid clearance from the brain and inability to inhibit parenchymal plasminogen activators. Together, these results suggest a novel therapy of infection-sensitized HI brain injury in newborns.

Plasminogen activator inhibitor-1 mitigates brain injury in a rat model of infection-sensitized neonatal hypoxia-ischemia.

Intrauterine infection exacerbates neonatal hypoxic-ischemic (HI) brain injury and impairs the development of cerebral cortex. Here we used low-dose lipopolysaccharide (LPS) pre-exposure followed by unilateral cerebral HI insult in 7-day-old rats to study the pathogenic mechanisms. We found that LPS pre-exposure blocked the HI-induced proteolytic activity of tissue-type plasminogen activator (tPA), but significantly enhanced NF-κB signaling, microglia activation, and the production of pro-inflammatory cytokines in newborn brains. Remarkably, these pathogenic responses were all blocked by intracerebroventricular injection of a stable-mutant form of plasminogen activator protein-1 called CPAI. Similarly, LPS pre-exposure amplified, while CPAI therapy mitigated HI-induced blood-brain-barrier damage and the brain tissue loss with a therapeutic window at 4 h after the LPS/HI insult. The CPAI also blocks microglia activation following a brain injection of LPS, which requires the contribution by tPA, but not the urinary-type plasminogen activator (uPA), as shown by experiments in tPA-null and uPA-null mice. These results implicate the nonproteolytic tPA activity in LPS/HI-induced brain damage and microglia activation. Finally, the CPAI treatment protects near-normal motor and white matter development despite neonatal LPS/HI insult. Together, because CPAI blocks both proteolytic and nonproteolytic tPA neurotoxicity, it is a promising therapeutics of neonatal HI injury either with or without infection.

Neurofibroma-associated macrophages play roles in tumor growth and response to pharmacological inhibition.

Neurofibromatosis type 1 (NF1) is a common genetic disease that predisposes 30-50 % of affected individuals to develop plexiform neurofibromas. We found that macrophage infiltration of both mouse and human neurofibromas correlates with disease progression. Macrophages accounted for almost half of neurofibroma cells, leading us to hypothesize that nerve macrophages are inflammatory effectors in neurofibroma development and/or growth. We tested the effects of PLX3397, a dual kit/fms kinase inhibitor that blocks macrophage infiltration, in the Dhh-Cre; Nf1(flox/flox) mouse model of GEM grade I neurofibroma. In mice aged 1-4 months, prior to development of nerve pathology and neurofibroma formation, PLX3397 did not impair tumor initiation and increased tumor volume compared to controls. However, in mice aged 7-9 months, after tumor establishment, a subset of mice demonstrating the largest reductions in macrophages after PLX3397 exhibited cell death and tumor volume regression. Macrophages are likely to provide an initial line of defense against developing tumors. Once tumors are established, they become tumor permissive. Macrophage depletion may result in impaired tumor maintenance and represent a therapeutic strategy for neurofibroma therapy.

CMV infection attenuates the disease course in a murine model of multiple sclerosis.

Recent evidence in multiple sclerosis (MS) suggests that active CMV infection may result in more benign clinical disease. The goal of this pilot study was to determine whether underlying murine CMV (MCMV) infection affects the course of the Theiler's murine encephalitis virus (TMEV) induced murine model of MS. A group of eight TMEV-infected mice were co-infected with MCMV at 2 weeks prior to TMEV infection while a second group of TMEV-infected mice received MCMV two weeks post TMEV. We also used 2 control groups, where at the above time points MCMV was replaced with PBS. Outcome measures included (1) monthly monitoring of disability via rotarod for 8 months; (2) in vivo MRI for brain atrophy studies and (3) FACS analysis of brain infiltrating lymphocytes at 8 months post TMEV infection. Co-infection with MCMV influenced the disease course in mice infected prior to TMEV infection. In this group, rotarod detectable motor performance was significantly improved starting 3 months post-infection and beyond (p≤0.024). In addition, their brain atrophy was close to 30% reduced at 8 months, but this was only present as a trend due to low power (p = 0.19). A significant reduction in the proportion of brain infiltrating CD3+ cells was detected in this group (p = 0.026), while the proportion of CD45+ Mac1+ cells significantly increased (p = 0.003). There was also a strong trend for a reduced proportion of CD4+ cells (p = 0.17) while CD8 and B220+ cell proportion did not change. These findings support an immunomodulatory effect of MCMV infection in this MS model. Future studies in this co-infection model will provide insight into mechanisms which modulate the development of demyelination and may be utilized for the development of novel therapeutic strategies.

Contrasting roles for CD4 vs. CD8 T-cells in a murine model of virally induced "T1 black hole" formation.

MRI is sensitive to tissue pathology in multiple sclerosis (MS); however, most lesional MRI findings have limited correlation with disability. Chronic T1 hypointense lesions or "T1 black holes" (T1BH), observed in a subset of MS patients and thought to represent axonal damage, show moderate to strong correlation with disability. The pathogenesis of T1BH remains unclear. We previously reported the first and as of yet only model of T1BH formation in the Theiler's murine encephalitis virus induced model of acute CNS neuroinflammation induced injury, where CD8 T-cells are critical mediators of axonal damage and related T1BH formation. The purpose of this study was to further analyze the role of CD8 and CD4 T-cells through adoptive transfer experiments and to determine if the relevant CD8 T-cells are classic epitope specific lymphocytes or different subsets. C57BL/6 mice were used as donors and RAG-1 deficient mice as hosts in our adoptive transfer experiments. In vivo 3-dimensional MRI images were acquired using a 7 Tesla small animal MRI system. For image analysis, we used semi-automated methods in Analyze 9.1; transfer efficiency was monitored using FACS of brain infiltrating lymphocytes. Using a peptide depletion method, we demonstrated that the majority of CD8 T-cells are classic epitope specific cytotoxic cells. CD8 T-cell transfer successfully restored the immune system's capability to mediate T1BH formation in animals that lack adaptive immune system, whereas CD4 T-cell transfer results in an attenuated phenotype with significantly less T1BH formation. These findings demonstrate contrasting roles for these cell types, with additional evidence for a direct pathogenic role of CD8 T-cells in our model of T1 black hole formation.

Long term antipsychotic treatment does not alter metabolite concentrations in rat striatum: an in vivo magnetic resonance spectroscopy study.

Proton magnetic resonance spectroscopy (MRS) studies of schizophrenic patients generally reveal reduced levels of N-acetyl aspartate (NAA) when compared with healthy controls. Whether this reduction is due to the disease or to the drugs used for treatment remains an open question. Numerous human and animal studies have attempted to determine the effects of antipsychotics on NAA levels with mixed results. The majority of the animal studies were ex vivo, which may not accurately reflect the in vivo situation, and limitations of the human studies include previous or concomitant medications or other confounds. To overcome these limitations, we dosed 10 rats/group for six months via drinking water with 0.2 or 2 mg/kg/day haloperidol or 10 or 30 mg/kg/day clozapine. Control rats received unadulterated water. Proton MRS data were collected longitudinally over the six month period from a 64 µL voxel containing primarily the right striatum prior to and monthly during drug administration and used to estimate the concentrations of NAA, creatine, and choline. Ratios of NAA, choline, inositol and glutamate+glutamine to creatine were also calculated. Only the Cho/Cr ratio showed a significant time-by-treatment effect (p=0.0285). These results are in agreement with previous studies of the striatum. However, regional and disease-specific effects remain unresolved.

Magnetic resonance imaging assessment of cardiac dysfunction in δ-sarcoglycan null mice.

Delta-sarcoglycan (δ-sarcoglycan) null, Scgd(-/-), mice develop cardiac and skeletal muscle histopathological alterations similar to those in humans with limb-girdle muscular dystrophy. The objective of this study was to assess the feasibility of using MRI to investigate cardiac dysfunction in Scgd(-/-) mice. Cardiac MRI of 8 month old Scgd(-/-) and wild type (WT) mice was performed. Compared to WT, Scgd(-/-) mice had significantly lower LV ejection fraction (44±5% vs. 66±4%, p=0.014), lower RV ejection fraction (25±2% vs. 51±3%, p<0.001) lower myocardial circumferential strain, (15.0±0.3% vs. 16.9±0.3%, p=0.007) and RV dilatation (54±3 µL vs. 40±3 µL, p=0.007). The regional circumferential strain also demonstrated significant temporal dyssynchrony between opposing regions of the Scgd(-/-) LV. Our results demonstrate severe cardiac dysfunction in Scgd(-/-) mice at 8 months. The study identifies a set of non-invasive markers that could be used to study efficacy of novel therapeutic agents in dystrophic mice.

Brain atrophy correlates with functional outcome in a murine model of multiple sclerosis.

White matter (WM) lesions are the classic pathological hallmarks of multiple sclerosis (MS). However, MRI-based WM lesion load shows relatively poor correlation with functional outcome, resulting in the "clinico-radiological paradox" of MS. Unlike lesion based measures, volumetric MRI assessment of brain atrophy shows a strong correlation with functional outcome, and the presence of early atrophy predicts a worse disease course. While extensive literature exists describing MRI characteristics of atrophy in MS, the exact pathogenesis and the substrate of atrophy-gray vs. WM loss, axonal/neuronal damage vs. demyelination, or a combination of the above-remain unclear. Animal models of atrophy would allow for detailed investigations of the pathomechanism, and would contribute to an enhanced understanding of structural-functional connections in this complex disease. We now report that in the Theiler's Murine Encephalitis Virus (TMEV) model of MS in SJL/J mice, significant brain atrophy accompanies the development of the progressive MS-like disease. We conducted volumetric MRI studies in 8 cases and 4 age, gender- and strain-matched control mice. While in controls we did not detect any brain atrophy, significant atrophy developed as early as 3 months into the disease course, and reached its peak by 6 months, resulting in ventricular enlargement by 118% (p=0.00003). A strong correlation (r=-0.88) between atrophy and disability, as assessed by rotarod assay, was also demonstrated. We earlier reported another neurodegenerative feature in this model, the presence of deep gray matter T2 hypointensity in thalamic nuclei. Future studies utilizing this model will allow us to investigate key components of MRI detectable neurodegenerative feature development, their tissue correlations and associations with functional outcome measures. These studies are expected to pave the way to a better understanding of the substrate of disability in MS models.

Ex vivo diffusion tensor imaging and neuropathological correlation in a murine model of hypoxia-ischemia-induced thrombotic stroke.

Diffusion tensor imaging (DTI) is a powerful method to visualize white matter, but its use in patients with acute stroke remains limited because of the lack of corresponding histologic information. In this study, we addressed this issue using a hypoxia-ischemia (HI)-induced thrombotic model of stroke in adult mice. At 6, 15, and 24 hours after injury, animals were divided into three groups for (1) in vivo T2- and diffusion-weighted magnetic resonance imaging, followed by histochemistry, (2) ex vivo DTI and electron microscopy, and (3) additional biochemical or immunochemical assays. The temporal changes of diffusion anisotropy and histopathology were compared in the fimbria, internal capsule, and external capsule. We found that HI caused a rapid reduction of axial and radial diffusivities in all three axonal bundles. A large decrease in fractional anisotropy, but not in axial diffusivity per se, was associated with structural breakdown of axons. Furthermore, the decrease in radial diffusivity correlated with swelling of myelin sheaths and compression of the axoplasma. The gray matter of the hippocampus also exhibited a high level of diffusion anisotropy, and its reduction signified dendritic degeneration. Taken together, these results suggest that cross-evaluation of multiple DTI parameters may provide a fuller picture of axonal and dendritic injury in acute ischemic stroke.

Therapeutic administration of plasminogen activator inhibitor-1 prevents hypoxic-ischemic brain injury in newborns.

Disruption of the integrity of the blood-brain barrier (BBB) is an important mechanism of cerebrovascular diseases, including neonatal cerebral hypoxia-ischemia (HI). Although both tissue-type plasminogen activator (tPA) and matrix metalloproteinase-9 (MMP-9) can produce BBB damage, their relationship in neonatal cerebral HI is unclear. Here we use a rodent model to test whether the plasminogen activator (PA) system is critical for MMP-9 activation and HI-induced brain injury in newborns. To test this hypothesis, we examined the therapeutic effect of intracerebroventricular injection of plasminogen activator inhibitor-1 (PAI-1) in rat pups subjected to unilateral carotid artery occlusion and systemic hypoxia. We found that the injection of PAI-1 greatly reduced the activity of both tPA and urokinase-type plasminogen activator after HI. It also blocked HI-induced MMP-9 activation and BBB permeability at 24 h of recovery. Furthermore, magnetic resonance imaging and histological analysis showed the PAI-1 treatment reduced brain edema, axonal degeneration, and cortical cell death at 24-48 h of recovery. Finally, the PAI-1 therapy provided a dose-dependent decrease of brain tissue loss at 7 d of recovery, with the therapeutic window at 4 h after the HI insult. Together, these results suggest that the brain PA system plays a pivotal role in neonatal cerebral HI and may be a promising therapeutic target in infants suffering hypoxic-ischemic encephalopathy.

Cerebral ischemia-hypoxia induces intravascular coagulation and autophagy.

Hypoxia is a critical factor for cell death or survival in ischemic stroke, but the pathological consequences of combined ischemia-hypoxia are not fully understood. Here we examine this issue using a modified Levine/Vannucci procedure in adult mice that consists of unilateral common carotid artery occlusion and hypoxia with tightly regulated body temperature. At the cellular level, ischemia-hypoxia produced proinflammatory cytokines and simultaneously activated both prosurvival (eg, synthesis of heat shock 70 protein, phosphorylation of ERK and AKT) and proapoptosis signaling pathways (eg, release of cytochrome c and AIF from mitochondria, cleavage of caspase-9 and -8). However, caspase-3 was not activated, and very few cells completed the apoptosis process. Instead, many damaged neurons showed features of autophagic/lysosomal cell death. At the tissue level, ischemia-hypoxia caused persistent cerebral perfusion deficits even after release of the carotid artery occlusion. These changes were associated with both platelet deposition and fibrin accumulation within the cerebral circulation and would be expected to contribute to infarction. Complementary studies in fibrinogen-deficient mice revealed that the absence of fibrin and/or secondary fibrin-mediated inflammatory processes significantly attenuated brain damage. Together, these results suggest that ischemia-hypoxia is a powerful stimulus for spontaneous coagulation leading to reperfusion deficits and autophagic/lysosomal cell death in brain.

High-resolution functional MRI at 3T in healthy and epilepsy subjects: hippocampal activation with picture encoding task.

Functional MRI (fMRI) studies of memory with coarse resolution of 4 x 4 x 5 mm often fail to demonstrate blood oxygenation level-dependent (BOLD) activation in the hippocampal formation. This failure occurs when nonactivating white matter is averaged with the signal from hippocampal gray matter, attenuating the total BOLD signal from a single voxel due to the "partial volume effect." In this study, we evaluated the suitability of high-resolution fMRI at 3T (voxel size 2 x 2 x 3 mm) for improved visualization of hippocampal activation during memory encoding in 21 healthy and 6 epilepsy subjects. We used a picture encoding task (block design) that involved memorization of indoor and outdoor scenes along with an appropriate resting task. Region-of-interest (ROI) analysis was performed; laterality indices (LIs) were calculated based on hippocampal ROIs (hROIs) or on global medial temporal ROIs (mtROIs). In 19 healthy subjects, robust bilateral BOLD signal changes within both ROIs were noted. The mean LI+/-SD for the hROIs is -0.12+/-0.06 and that for the medial temporal ROIs -0.12+/-0.05, with correlation between the LIs (r = 0.59, P = 0.009). Good concordance was noted between the surgical outcome and memory lateralization with the fMRI task employed in this study. The preliminary results are encouraging, and with continuing improvements in MRI scanner technology, we expect fMRI of the hippocampal formation at higher resolution to be possible and preferable. Furthermore, these results suggest that a larger study to test the utility of high-resolution fMRI in epilepsy presurgical evaluation is needed.

Practical aspects of conducting large-scale functional magnetic resonance imaging studies in children.

The potential benefits of functional magnetic resonance imaging (MRI) for the investigation of normal development have been limited by difficulties in its use with children. We describe the practical aspects, including failure rates, involved in conducting large-scale functional MRI studies with normal children. Two hundred and nine healthy children between the ages of 5 and 18 years participated in a functional MRI study of language development. Reliable activation maps were obtained across the age range. Younger children had significantly higher failure rates than older children and adolescents. It is concluded that it is feasible to conduct large-scale functional MRI studies of children as young as 5 years old. These findings can be used by other research groups to guide study design and plans for recruitment of young subjects.