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Blood-brain barrier hyperpermeability precedes demyelination in the cuprizone model.

ABSTRACT: In neuroinflammatory disorders such as multiple sclerosis, the physiological function of the blood-brain barrier (BBB) is perturbed, particularly in demyelinating lesions and supposedly secondary to acute demyelinating pathology. Using the toxic non-inflammatory cuprizone model of demyelination, we demonstrate, however, that the onset of persistent BBB impairment precedes demyelination. In addition to a direct effect of cuprizone on endothelial cells, a plethora of inflammatory mediators, which are mainly of astroglial origin during the initial disease phase, likely contribute to the destabilization of endothelial barrier function in vivo. Our study reveals that, at different time points of pathology and in different CNS regions, the level of gliosis correlates with the extent of BBB hyperpermeability and edema. Furthermore, in mutant mice with abolished type 3 CXC chemokine receptor (CXCR3) signaling, inflammatory responses are dampened and BBB dysfunction ameliorated. Together, these data have implications for understanding the role of BBB permeability in the pathogenesis of demyelinating disease.

SUBMITTER: Berghoff SA

PROVIDER: S-EPMC5710130 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Blood-brain barrier hyperpermeability precedes demyelination in the cuprizone model.

Berghoff Stefan A SA Düking Tim T Spieth Lena L Winchenbach Jan J Stumpf Sina K SK Gerndt Nina N Kusch Kathrin K Ruhwedel Torben T Möbius Wiebke W Saher Gesine G

Acta neuropathologica communications 20171201 1

In neuroinflammatory disorders such as multiple sclerosis, the physiological function of the blood-brain barrier (BBB) is perturbed, particularly in demyelinating lesions and supposedly secondary to acute demyelinating pathology. Using the toxic non-inflammatory cuprizone model of demyelination, we demonstrate, however, that the onset of persistent BBB impairment precedes demyelination. In addition to a direct effect of cuprizone on endothelial cells, a plethora of inflammatory mediators, which ...[more]

PMID: 29195512

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Project description:The development of new neurotherapeutics depends on appropriate animal models being chosen in preclinical studies. The cuprizone model is an effective tool for studying demyelination and remyelination processes in the brain, but blood-brain barrier (BBB) integrity in the cuprizone model is still a topic for debate. Several publications claim that the BBB remains intact during cuprizone-induced demyelination; others demonstrate results that could explain the increased BBB permeability. In this study, we aim to analyze the permeability of the BBB for different macromolecules, particularly antibody conjugates, in a cuprizone-induced model of demyelination. We compared the traditional approach using Evans blue injection with subsequent dye extraction and detection of antibody conjugates using magnetic resonance imaging (MRI) and confocal microscopy to analyze BBB permeability in the cuprizone model. First, we validated our model of demyelination by performing T2-weighted MRI, diffusion tensor imaging, quantitative rt-PCR to detect changes in mRNA expression of myelin basic protein and proteolipid protein, and Luxol fast blue histological staining of myelin. Intraperitoneal injection of Evans blue did not result in any differences between the fluorescent signal in the brain of healthy and cuprizone-treated mice (IVIS analysis with subsequent dye extraction). In contrast, intravenous injection of antibody conjugates (anti-GFAP or non-specific IgG) after 4 weeks of a cuprizone diet demonstrated accumulation in the corpus callosum of cuprizone-treated mice both by contrast-enhanced MRI (for gadolinium-labeled antibodies) and by fluorescence microscopy (for Alexa488-labeled antibodies). Our results suggest that the methods with better sensitivity could detect the accumulation of macromolecules (such as fluorescent-labeled or gadolinium-labeled antibody conjugates) in the brain, suggesting a local BBB disruption in the demyelinating area. These findings support previous investigations that questioned BBB integrity in the cuprizone model and demonstrate the possibility of delivering antibody conjugates to the corpus callosum of cuprizone-treated mice.

Project description:The main objective of this study was to determine the cellular and molecular effects of doxycycline on the blood-brain barrier (BBB) and protection against secondary injuries following traumatic brain injury (TBI). Microvascular hyperpermeability and cerebral edema resulting from BBB dysfunction after TBI leads to elevation of intracranial pressure, secondary brain ischemia, herniation, and brain death. There are currently no effective therapies to modulate the underlying pathophysiology responsible for TBI-induced BBB dysfunction and hyperpermeability. The loss of BBB integrity by the proteolytic enzyme matrix metalloproteinase-9 (MMP-9) is critical to TBI-induced BBB hyperpermeability, and doxycycline possesses anti-MMP-9 effect. In this study, the effect of doxycycline on BBB hyperpermeability was studied utilizing molecular modeling (using Glide) in silico, cell culture-based models in vitro, and a mouse model of TBI in vivo. Brain microvascular endothelial cell assays of tight junction protein immunofluorescence and barrier permeability were performed. Adult C57BL/6 mice were subjected to sham versus TBI with or without doxycycline treatment and immediate intravital microscopic analysis for evaluating BBB integrity. Postmortem mouse brain tissue was collected to measure MMP-9 enzyme activity. It was found that doxycycline binding to the MMP-9 active sites have binding affinity of -7.07 kcal/mol. Doxycycline treated cell monolayers were protected from microvascular hyperpermeability and retained tight junction integrity (p < 0.05). Doxycycline treatment decreased BBB hyperpermeability following TBI in mice by 25% (p < 0.05). MMP-9 enzyme activity in brain tissue decreased with doxycycline treatment following TBI (p < 0.05). Doxycycline preserves BBB tight junction integrity following TBI via inhibiting MMP-9 activity. When established in human subjects, doxycycline, may provide readily accessible medical treatment after TBI to attenuate secondary injury.

Project description:Recent research has revealed that uncontrolled chronic neuroinflammation is closely associated with diverse neurodegenerative diseases, by impairing blood-brain barrier (BBB) function and astrocytic reaction. Endoplasmic reticulum (ER) stress is conventionally linked to the loss of neuronal structure and function and should be widely attenuated. This notion has been questioned by recent experimental studies, which have shown that non-harmful levels of ER stress had numerous beneficial roles against neurodegeneration, including neuroprotection and inhibition of cytokine production. Here, we investigated the mild ER stress-based regulation of LPS-induced inflammatory responses in astrocytes. Primary astrocytes were exposed to tunicamycin (TM), a compound that activates ER stress, with or without the ER-stress inhibitor sodium 4-phenylbutyrate (4-PBA) before LPS treatment. Astrocytic activation, proinflammatory factor production, and the extent of ER stress were assessed. In addition, the effect of mild ER stress on astrocytes and BBB function was determined in vivo. Male Sprague-Dawley rats received intracerebroventricular injections of TM with or without intraperitoneal 4-PBA before LPS administration. The levels of astrocytic activation and BBB permeability were measured after treatment. Our results showed that lower doses of TM resulted in a mild ER-stress response without inducing cytotoxicity and tissue toxicity. Non-toxic ER-stress preconditioning ameliorated LPS-induced overactivation and inflammatory responses in astrocytes. Moreover, pre-exposure to non-lethal doses of TM improved LPS-induced BBB impairment and cognitive ability dysfunction in rats. However, 4-PBA, reversed the protective effect of TM preconditioning in vitro and in vivo. We conclude that mild ER stress ("preconditioning") can alleviate LPS-induced astrocytic activation and BBB disruption. Our findings provide a better understanding for the regulatory role of ER stress in neuroinflammation and indicate that mild ER stress might have therapeutic value for the treatment of neurodegenerative diseases.

Project description:BACKGROUND:Various studies by MRI exhibit that the corpus callosum (CC) is the most vulnerable to cuprizone administration, detecting the demyelination and remyelination process using different MRI parameters are, however, lacking. PURPOSE:To investigate the sensitivity of multiparametric MRI both in vivo and ex vivo for demyelination and remyelination. STUDY TYPE:Prospective. ANIMAL MODEL:A cuprizone mice model with an age-matched control group (n = 5), 4-week cuprizone exposure group followed by 9-week on a normal diet (n = 6), and a 13-week cuprizone exposure group (n = 6). FIELD STRENGTH/SEQUENCE:3D gradient recalled echo, T2 -weighted, and diffusion tensor imaging (DTI) at 7.0T and 9.4T. ASSESSMENT:Quantification of DTI metrics, quantitative susceptibility mapping (QSM), and T2 -weighted imaging intensity in major white matter bundles. STATISTICAL TESTS:Nonparametric permutation tests were used with a cluster-forming threshold as 3.09 (equivalent to P = 0.001), and the significant level as P = 0.05 with family-wise correction. RESULTS:In vivo susceptibility values increased from -11.7 to -0.7 ppb (P < 0.001) in CC and from -13.7 to -5.1 ppb (P < 0.001) in the anterior commissure (AC) after the 13-week cuprizone exposure. Ex vivo susceptibility values increased from -25.4 to 7.4 ppb (P < 0.001) in CC and from -41.6 to -15.8 ppb (P < 0.001) in AC. Susceptibility values showed high variations to demyelination for in vivo studies (94.0% in CC, 62.8% in AC). Susceptibility values exhibited higher variations than radial diffusivity for ex vivo studies (129.1% vs. 28.3% in CC, 62.0% vs. 25.0% in AC). In addition to the differential susceptibility variations in different white matter tracts, intraregional demyelination variation was also present not only in CC but also in the AC area by voxel-based analysis. DATA CONCLUSION:QSM is sensitive to the demyelination process of cuprizone exposure, which can be a complementary technique to conventional T2 -weighted images and DTI metrics. LEVEL OF EVIDENCE:2 Technical Efficacy Stage: 2 J. Magn. Reson. Imaging 2019;50:1852-1865.

Dataset Information

Blood-brain barrier hyperpermeability precedes demyelination in the cuprizone model.

Publications

Blood-brain barrier hyperpermeability precedes demyelination in the cuprizone model.

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