Astroglial NF-kB contributes to white matter damage and cognitive impairment in a mouse model of vascular dementia
© The Author(s). 2016
Received: 15 July 2016
Accepted: 19 July 2016
Published: 4 August 2016
Vascular cognitive impairment is the second most common form of dementia. The pathogenic pathways leading to vascular cognitive impairment remain unclear but clinical and experimental data have shown that chronic reactive astrogliosis occurs within white matter lesions, indicating that a sustained pro-inflammatory environment affecting the white matter may contribute towards disease progression. To model vascular cognitive impairment, we induced prolonged mild cerebral hypoperfusion in mice by bilateral common carotid artery stenosis. This chronic hypoperfusion resulted in reactive gliosis of astrocytes and microglia within white matter tracts, demyelination and axonal degeneration, consecutive spatial memory deficits, and loss of white matter integrity, as measured by ultra high-field magnetic resonance diffusion tensor imaging. White matter astrogliosis was accompanied by activation of the pro-inflammatory transcription factor nuclear factor (NF)-kB in reactive astrocytes. Using mice expressing a dominant negative inhibitor of NF-kB under the control of the astrocyte-specific glial fibrillary acid protein (GFAP) promoter (GFAP-IkBα-dn), we found that transgenic inhibition of astroglial NF-kB signaling ameliorated gliosis and axonal loss, maintained white matter structural integrity, and preserved memory function. Collectively, our results imply that pro-inflammatory changes in white matter astrocytes may represent an important detrimental component in the pathogenesis of vascular cognitive impairment, and that targeting these pathways may lead to novel therapeutic strategies.
Vascular cognitive impairment (VCI) is a common neurodegenerative disease characterised by impaired cognitive function attributable to cerebrovascular pathology . In its pure form, VCI is the second most common form of dementia and represents an important co-factor for the development of Alzheimer’s disease and other neurodegenerative disorders . Neuropathologically, VCI is characterised by demyelination and axonal loss within white matter tracts , which has traditionally been attributed to the direct effects of chronic oxygen deprivation. However, several lines of evidence suggest that chronic neuroinflammation may also play a detrimental role in the initiation and progression of VCI. First, cerebrovascular and cardiovascular diseases, which are risk factors for VCI, induce a chronic pro-inflammatory environment, particularly in and around blood vessels [4, 5]. Second, increased levels of pro-inflammatory markers have been detected within white matter lesions in animal models and post-mortem studies of VCI patients [6, 7], and alterations of blood–brain barrier integrity are a central and perhaps aggravating element of VCI [8, 9]. Finally, in patients and animal models of VCI, reactive gliosis of microglia and astrocytes is present within the white matter [10, 11]. Reactive astrogliosis is a pervasive defence mechanism in response to cerebral injury that may promote neurodegeneration or neuroprotection in a context-dependent manner . However, the roles of astrogliosis and the various inflammatory cascades mediated by reactive astrocytes in chronic cerebral hypoperfusion and VCI remain poorly understood.
Here, we have used an animal model that recapitulates many of the neuropathological and behavioural hallmarks of VCI . We demonstrate that reactive astrogliosis induces activation of the pro-inflammatory nuclear factor (NF)-kB pathway in astrocytes, and that transgenic inhibition of this pathway confers protection against axonal loss, helps to promote white matter integrity, and preserves cognitive function.
Materials and methods
All experiments were approved by a local animal welfare committee. We used 10–14 week-old male GFAP-IkBα-dn mice  and their wildtype age-matched male littermates. Animals were housed in groups (two to five per cage) on a 12 h light/dark cycle with food and water available ad libitum.
Chronic hypoperfusion model
Gold plated stainless steel microcoils (Sawane) were wrapped around both common carotid arteries in isoflurane-anaesthetised mice . Informed by earlier studies that revealed either subtle [15, 16] or very pronounced changes , we modified the existing model by using custom-made coils with an internal diameter of 170 μm. Animals were anaesthetised with isoflurane (1.5 % v/v) and maintained on a heating pad (37 °C). The common carotid arteries were exposed through a midline cervical incision, and the microcoils were wrapped around both common carotid arteries. A 30 min interval was allowed between each microcoil insertion. Sham-treated animals underwent identical procedures except that coils were not implanted. Local lidocaine gel and intraperitoneal buprenorphine were applied as analgesics. Mice from each litter were randomised to either microcoil insertion or sham surgery. All experiments and subsequent data analysis were conducted in a blinded manner, with respect to genotype and treatment. Mice were excluded if visual damage to the carotid arteries or bleeding during surgery occurred. In addition, carotid arteries were inspected for patency at the end of experiments, and animals with occluded carotid arteries were also excluded.
Laser speckle perfusion imaging and PcomA assessment
Animals were anaesthetised with isoflurane (1.5 % v/v), and the skull was exposed through a midline incision. A cranial window was created and closed by gluing a glass coverslip onto the skull and adhering the skin to the coverslip rim. For imaging, mice were anaesthetised with isoflurane (1 % v/v) and fixed within a stereotaxic frame (Narishige). Brain perfusion was determined through a closed cranial window in anaesthetised mice using PeriCam PSI Laser Speckle Imager and PIMsoft software (Perimed). PcomA development was determined by transcardial perfusion with India ink (Pelikan; 10 % in water containing 10 % gelatine), followed by decapitation and fixation in 4 % paraformaldehype (PFA). PcomA was scored as: 0, absent; 1, hypoplastic; 2, truncal. A single PcomA score was calculated by averaging left and right scores.
Ultra high-field MRI
MRI of isoflurane-anaesthetised mice was performed on an 11.7 T horizontal small-bore magnet (Biospec 117/16, Bruker) using a two-element transmit/receive proton (1H) cryocoil (Bruker Biospin). Body temperature was maintained at 37 °C via an integrated water heating system. Respiratory rate was maintained at 70-80 cycles/min. Anatomical images were acquired using a rapid acquisition relaxation enhancement (RARE) T2-weighted (T2-w) sequence [echo time (TE) = 0.01 s; repetition time (TR) = 2.5 s; in-plane resolution 45 μm2], and a RARE T1-weighted (T1-w) sequence (TE/TR = 0.01 s/1 s; in-plane resolution 45 μm2). White matter integrity was assessed using an EPI DTI sequence (TE/TR 26/4000 ms; b value 1000 s/mm2; 60 diffusion directions; 5 b0 images; in-plane resolution 100 μm2).
Coronal sections (20 μm) were obtained from PFA-fixed hemispheres, blocked with 10 % normal goat serum (Vector Labs) and 0.3 % Triton-X100 (Sigma) in PBS for 1 h, and incubated with primary antibodies in PBS containing 5 % goat serum and 0.05 % Triton overnight at 4 °C. The following primary antibodies were used: rabbit anti-GFAP (1:1000; Z0334, Dako), rat anti-GFAP (1:1000; 13-0300, Invitrogen), rabbit anti-Iba1 (1:500; #019-19741, Wako), mouse anti-SMI312 (1:500, smi-312r, Covance), rabbit anti-p65 (1:100; sc-372, Santa Cruz; or #3033, Cell Signaling). Stainings were visualised using secondary antibodies from goat conjugated with Alexa Fluor 488, 596 or 647 (1:1000; Invitrogen; 2 h at room temperature). Nuclei were stained with Hoechst 33258 (1:1000; Invitrogen). Omission of primary antibodies served as negative controls. Confocal images were acquired using a confocal laser scanning microscope (LSM 700; Zeiss).
Cytokine and NF-kB quantification
Mice were sacrificed, and the corpus callosum was dissected and stored in liquid nitrogen. Samples were homogenised in PBS containing protease/phosphatase inhibitor (Thermo Scientific) using a Precellys 24 homogeniser (Peqlab), and lysed in RIPA buffer (25 mM TRIS, 150 mM NaCl, 1 % NP-40, 0.5 % Na-deoxycholate, 0.1 % SDS, pH 7.2). Quantitative cytokine determination was performed using an electrochemiluminescence ELISA (Mouse ProInflammatory Ultra-Sensitive Kit, Meso Scale Discovery). Signals were measured on a SECTOR Imager 2400 reader (Meso Scale Discovery). For NF-kB measurements, nuclear extracts were created from whole brain homogenates (Nuclear Extraction Kit, #2900, Millipore), and NF-kB activity in nuclear extracts was determined with a commercial assay (EZ-TFA assay, #70-610, Millipore) according to the manufacturer’s instructions using a microplate reader (FLUOstar Omega, BMG Labtech). All measurements were performed in duplicates.
Male GFAP-IkBα-dn and wildtype mice have similar phenotypes in behavioural paradigms, including spatial learning and memory . All tests were recorded and analysed using Ethovision XT9 (Noldus). The Y maze consisted of three arms (length, 34.5 cm; width, 8 cm; height, 12.5 cm) extruding at equal angles from a central platform. The Y Maze test was modified from previous designs . During the acquisition trial, one arm was closed and mice were placed at the end of an open arm (chosen at random) and allowed to explore the two open arms for 5 min. After 24 h, mice were returned to the maze with all 3 arms open; the start point corresponding to the individual start point of each mouse in the acquisition trial, and allowed to explore for 5 min. Arm preference was quantified as the ratio between the time spent in one arm and the time spent in all arms (preferency index). For open field exploration, mice were placed in the centre of a dimly lit (20–30 lx) arena. The area was virtually divided into a centre and an area ≤5 cm to the walls (surround). General locomotor activity was quantified as total distance moved and velocity. The level of anxiety was estimated by the percentage of time spent in the surround.
Immunohistochemical images were imported into ImageJ 1.50 (W. Rasband, NIH), smoothed and despeckled using a median filter, and converted to an 8-bit grey level image. Following contrast enhancement (0.4 % saturated pixels), images were binarised with a threshold defined as the mean background intensity plus the standard deviation of background intensity multiplied by 3. Astrocyte area coverage and diameter, and micoglial cell count were determined automatically by ImageJ. For astrocyte process length, binarised images were skeletonised and analysed using the Analyze Skeleton (2D/3D) plugin for ImageJ. These semi-automatic quantifications were compared with a subset of data (n = 3 from each group) that was quantified by manual analysis; no significant difference was detected between the analysis methods (data not shown). GFAP-positive astrocytes were considered positive for p65 when they showed a spherical somatic p65 staining that was smaller than the GFAP-positive cell soma and overlapped with the Hoechst 33258 nuclear counterstain. Mean pixel intensity of SMI312 staining in 8-bit grey level images was taken as a measure of axonal integrity. Demyelination was analysed in LFB-stained sections using a semi-quantitative scoring system (0 = no demyelination; 1 = visible fiber disarrangement; 2 = vacuoles and focal demyelination; 3 = widespread/continuous demyelination). Examples are provided in Additional file 1: Figure S1. Analysis of the corpus callosum was performed within pre-defined areas (0.75 mm and 2.0 mm from the midline). All measurements were taken in triplicates, and the values averaged. Laser speckle contrast data were down-sampled, exported into Excel (Microsoft), and normalised to perfusion levels before microcoil implantation. Paravision 5.1 (Bruker) was used to generate the parametric images used for white matter analysis. Fractional anisotropy (FA), apparent diffusion coefficient (ADC), axial diffusivity (AD) and radial diffusivity (RD) were measured. Direction-encoded colour (DEC) maps based on the primary eigenvectors were generated using in-house software. The colour determines the principal direction (X, Y or Z) of the primary eigenvector exhibiting the largest diffusion coefficient from the measured diffusion tensor: red designates medial to lateral orientation, blue represents dorsal to ventral orientation and green exhibits the rostral to caudal orientation. Regions of interest (ROIs) including the corpus callosum, external capsule and internal capsule were manually drawn onto the diffusion tensor images, and verified by comparison with the corresponding T2-w image and a stereotaxic mouse brain atlas . Heatmaps were generated by a) registering the FA images to a reference image within the respective cohort and b) setting landmarks on trace-weighted diffusion images and transferring them onto their respective FA images. Registration was performed using the bUnwarp plugin (version 2.6.3) for ImageJ.
Comparisons between two groups were conducted using the Mann–Whitney test. Differences between several groups were analysed using the Kruskal-Wallis test followed by Dunn’s multiple comparisons test. Data were analysed using Prism 6 (GraphPad), and are expressed as mean ± SEM; p < 0.05 was accepted as statistically significant.
Gliosis, demyelination, axonal loss and memory deficits in a mouse model of VCI
Moreover, we found that the concentrations of the pro-inflammatory cytokines, interleukin-1 and interleukin-6, were increased in homogenates of the corpus callosum isolated from mice subjected to BCAS (Fig. 1j).
NF-kB is activated by chronic hypoperfusion in reactive white matter astrocytes
One consequence of the expression of pro-inflammatory cytokines such as IL-1 is the activation of the nuclear factor (NF)-kB signaling cascade in reactive astrocytes . Under control conditions, inactive NF-kB is localised to the cytoplasm by its interaction with inhibitory IkB proteins such as IkBα. Dissociation from IkB and subsequent nuclear translocation of NF-kB is initiated by the degradation of IkB through cytokine-induced IkB kinase activation . Once activated, NF-kB acts as an important regulator of reactive gliosis, inflammation, myelination and neuronal survival in different disease models [12, 14, 23].
Astrocyte-specific transgenic inhibition of NF-kB activity confers protection against structural and cognitive decline following chronic hypoperfusion
To investigate the consequences of astroglial NF-kB activation, we used transgenic GFAP-IkBα-dn mice in which astroglial NF-kB activation is selectively inhibited by targeting the overexpression of a dominant negative form of IkBα to astrocytes . Earlier studies have shown that the transgene is selectively active in astrocytes, while NF-kB activity remains unchanged in neurons . Following BCAS, we found that nuclear p65 was absent from reactive white matter astrocytes (Fig. 3d) and that NF-kB activity was similar to sham-treated mice (Fig. 3c).
Interestingly, astrogliosis was detectable in GFAP-IkBα-dn mice following BCAS but reactive microglial activation and demyelination in white matter structures were significantly attenuated (Fig. 4c-e). Furthermore, axonal integrity within the corpus callosum was preserved in GFAP-IkBα-dn mice compared with sham-treated controls (Fig. 4f). Importantly, GFAP-IkBα-dn mice subjected to hypoperfusion spent significantly more time in the novel arm in the retrieval trial of the Y maze test (Fig. 4g), indicating preserved spatial memory function as a consequence of decreased NF-kB signaling. The open field test revealed no differences in distance moved, velocity or time spent in the surround between hypoperfused GFAP-IkBα-dn, sham-treated GFAP-IkBα-dn and wildtype mice (Fig. 4h-j). Overall, these data show that NF-kB activation in reactive astrocytes contributes to neuropathological damage and behavioural changes in our VaD model.
Ultra high-field diffusion tensor imaging reveals preserved structural integrity of white matter tracts in GFAP-IkBα-dn mice following BCAS
In this study, we investigated the role of the pro-inflammatory NF-kB pathway in reactive astroglia in a mouse model of vascular cognitive impairment and found that transgenic inhibition of NF-kB signaling confers protection from axonal loss, gliosis, demyelination, deterioration of white matter integrity and memory impairment.
Although vascular cognitive impairment is associated with an enormous clinical and socioeconomic burden, causal treatment options are absent, mostly because the molecular pathways triggered by chronic cerebral hypoperfusion remain poorly understood. Importantly, clinical and experimental studies have shown that vascular impairment is associated with inflammatory changes and reactive gliosis of the white matter [4–7, 9], indicating that white matter damage is not simply a consequence of chronic oxygen deprivation but is induced and sustained by a pro-inflammatory environment. The hallmarks of chronic inflammation:reactive gliosis of white matter astrocytes and microglia, and expression of pro-inflammatory cytokines, were also detected in our model and associated with axonal degeneration, demyelination, spatial memory impairment and loss of white matter integrity. Importantly, the DTI signatures of hypoperfused mice corroborated with the demyelination and axonal degeneration observed on histology , and were comparable to those observed in clinical vascular cognitive impairment . The cellular sources of IL-1 and IL-6 are difficult to define in vivo using our model. However, earlier studies have shown that microglia and, to a lesser degree, astrocytes release these cytokines during hypoxia [1, 23, 28]. Hence, both cell types may induce and sustain the pro-inflammatory environment that ultimately leads to demyelination. Moreover, progressive demyelination may further exacerbate gliosis .
Reactive astrogliosis is a pervasive but insufficiently understood response mechanism of the brain to acute or chronic injury . Interestingly, we found that astrogliosis induces activation of the pro-inflammatory NF-kB pathway, and that transgenic inhibition of NF-kB signaling in reactive astrocytes confers protection from neurodegeneration and cognitive impairment. Notably, some degree of gliosis was evident in transgenic mice after BCAS, indicating that multiple pathways may be involved . The protective effect of NF-kB on white matter integrity was also detectable using DTI, underlining the significance of DTI as a longitudinal marker of treatment response in future translational studies.
It should be noted that the model used in the present study, and arguably in previously published animal studies thus far , does not reflect certain aspects of clinical vascular cognitive impairment including the chronic evolution of the disease over decades, the predominant affection of small vessels, and cardiovascular and environmental co-morbidities. Therefore, an important question for future studies is whether the protective effect of NF-kB inhibition is evident in models that recapitulate other aspects of human vascular cognitive impairment. One other important point to address will be whether astroglial NF-kB contributes to pathways such as those mediated by hypoxia-inducible transcription factor-1 activation or perturbed glutamate uptake [29, 30] in models of vascular cognitive impairment. Furthermore, as NF-kB pathway inhibitors progress through pre-clinical development , it will be of great interest to investigate whether these compounds confer comparable protection from chronic hypoxia.
The NF-kB pathway has been implicated in other pathological conditions such as autoimmune or toxic demyelination and spinal cord injury [14, 23, 32]. Here, we demonstrated that activation of NF-kB in astrocytes contributes not only to white matter demyelination and axonal loss during chronic hypoxia but that an astrocytic pro-inflammatory pathway may have important consequences for cognitive outcome in neurodegenerative disease.
Our study has demonstrated that chronic activation and gliosis of astrocytes are central components of disease pathogenesis in a model of vascular cognitive impairment, and that therapeutic strategies targeting these pathways may lead to novel treatment options for vascular dementia.
DTI, Diffusion tensor imaging; GFAP, Glial fibrillary acid protein; MRI, Magnetic resonance imaging; NF-kB, Nuclear factor kappa-light-chain-enhancer of activated B cells; VCI, Vascular cognitive impairment
We thank John Bethea (Miami) for providing the transgenic mice. We are grateful to Jan Peter and Theresa Hundt (Bonn) for help with immunohistochemistry, biochemical experiments and genotyping. We thank Marco Foddis (Berlin) for help with establishing the BCAS model, Manuel Schölling and Christoph Moehl (Bonn) for help with image analysis, and Michael Heneka (Bonn) for help with electrochemiluminescence measurements.
This work was supported by grants to GCP from the Network Of Centres Of Excellence In Neurodegeneration (CoEN), EU Joint Programme—Neurodegenerative Disease Research (JPND) program (EU Horizon 2020 research and innovation program, grant agreement No 643417/DACAPO-AD), and the DZNE.
Availability of data and material
The paper does not include proprietary material or any data for which deposition in a public repository is mandatory, but all relevant data can be made available from the corresponding author upon request.
RS, TS and GCP implemented the methodology and designed the experiments. RS developed and optimised the MRI sequences. RS, TS, FG, CR, KT and AD performed the experiments. RS, TS, FG and GCP analysed the data. RS and GCP wrote the manuscript. All authors discussed the results and commented on the manuscript at all stages. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
All experiments were performed according to animal care guidelines and approved by a local animal welfare committee.
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