Dickkopf-1 accentuates neuronal damage upon multifocal cerebral microinfarctions by impairing vascular and immune responses

Dickkopf-1 accentuates neuronal damage upon multifocal cerebral microinfarctions by impairing vascular and immune responses | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Dickkopf-1 accentuates neuronal damage upon multifocal cerebral microinfarctions by impairing vascular and immune responses Esther Trudel, Anne-Sophie Allain, Ayman ElAli This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7622603/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 06 Jan, 2026 Read the published version in Discover Neuroscience → Version 1 posted 11 You are reading this latest preprint version Abstract Cerebral small vessel disease (cSVD) associated with the occlusion of small penetrating arterioles leading to multifocal microinfarcts, constitutes common microangiopathies affecting brain functions. Dickkopf-1 (DKK1) is a potent inhibitor of the canonical Wnt pathway, which plays key roles in regulating neurovascular and immune functions. DKK1 levels are reported to be increased in cerebrovascular diseases, correlating with poor prognosis. Herein, we aimed to investigate the contribution of DKK1 elevated levels to the pathobiology of cSVD associated with microinfarcts. This was achieved using a transgenic mouse model that enables a conditional tissue-specific induction of DKK1. cSVD was induced via intravascular injection of micro-emboli to occlude penetrating arterioles. Our results indicate that the circulating levels of endogenous DKK1 are increased after cSVD and remain steadily elevated before progressively returning to basal levels. DKK1 conditional induction with respect to its temporal regulation after cSVD exacerbates vascular permeability and alters immediate cerebrovascular reactivity, outlining deregulation of the neurovascular functions. Furthermore, DKK1 induction promotes progressive neuronal loss and impairs neurogenesis after cSVD. We show that DKK1 elevated levels are associated with attenuation of myeloid cell recruitment to the lesion sites, accompanied with a persistent polarization of monocytes in the blood circulation towards a pro-inflammatory phenotype. Notably, we reveal that DKK1 elevated levels for a prolonged period after cSVD mediates pathological changes that remain persistent despite delayed normalization of its expression. Our study suggests that circulating DKK1 decisively affects the pathobiology of cSVD associated with microinfarcts, outlining its potential as a prognostic marker and a therapeutic target. Dickkopf-1 cerebral small vessel disease (cSVD) microinfarcts neurovascular functions monocytes inflammation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Cerebral small vessel disease (cSVD) encompasses a diverse group of microangiopathies and represents the most common chronic and progressive cerebrovascular disease [ 1 ]. cSVD affects nearly 80% of people over the age of 60, accounts for about 25% of ischemic stroke, often occurs with Alzheimer’s disease (AD), and is now recognized as a major contributor to vascular dementia (VaD) [ 2 – 4 ]. The neuropathological features of cSVD are heterogenous and include subcortical microinfarcts, lacunar infarcts, microbleeds, enlarged perivascular spaces, white matter hyperintensities (WMH), and brain atrophy [ 5 ]. Depending upon the affected brain structure, distinct as well as common clinical manifestations occur, including stroke-related symptoms, cognitive decline, dementia, and psychiatric disorders [ 6 , 7 ]. The prevalence of cSVD is expected to continue rising over the next decade due to the ageing population and the high incidence of various vascular risk factors, including metabolic diseases, thereby imposing a significant socioeconomic burden [ 8 , 9 ]. A better understanding of the mechanisms underlying the pathobiology of cSVD could improve patient management upon hospitalization and support the development of new effective preventative and therapeutic interventions. cSVD associated with occlusion of small penetrating arterioles, leading to multifocal microinfarcts, represents a very common type [ 10 ]. The pathobiological processes essentially converge towards deregulation of neurovascular functions, inflammation, and secondary neurodegeneration [ 11 , 12 ]. Dickkopf-1 (DKK1) is a secreted glycoprotein that act as a potent inhibitor of the canonical Wnt pathway, which plays a major role in regulating neurovascular and immune functions [ 13 – 15 ]. While DKK1 expression is absent in the healthy brain, it becomes detectable under various vascular-related pathological conditions, including ischemia [ 14 , 16 ]. DKK1 increased expression narrowly correlates with deregulation of canonical Wnt pathway activity [ 14 , 17 , 18 ]. DKK1 is abundantly expressed in the blood circulation, presumably deriving from the bone marrow, where it plays an important role in regulating hematopoiesis [ 19 ]. Notably, DKK1 levels are elevated in the blood circulation of patients with acute and stable cerebrovascular diseases associated with large- as well as small-artery occlusion (SAO), correlating with poor prognosis [ 20 – 23 ]. Furthermore, DKK1 circulating levels are reported to be elevated in most of the leading risk factors of cSVD, mainly hypertension, hyperlipidemia, cardiovascular diseases and metabolic disorders [ 16 , 24 – 28 ]. These observations suggest that DKK1 is potentially involved in modulating the pathobiology of cSVD associated with microinfarcts. Herein, we aimed to investigate the role of DKK1 in affecting the pathobiology of cSVD associated with microinfarcts. This was achieved using a novel transgenic mouse model that enables a conditional widespread induction of DKK1 in a tissue-specific manner (i.e. inducible DKK1 mice). cSVD associated with microinfarcts was induced via sporadic micro-occlusion (MO) of cerebral penetrating arterioles using intravascularly injected microbeads (i.e. micro-emboli). Herein, we show that endogenous DKK1 levels immediately increase in the blood circulation following cSVD, remain elevated for several weeks, and return to basal levels at one month, outlining a prolonged, but transient upregulation. Our results reveal that DKK1 conditional widespread induction in respect to its endogenous temporal regulation after cSVD worsens vascular permeability as well as alters vascular reactivity translated by an impaired acute brain perfusion after micro-occlusions. These changes are associated with aggravated neuronal loss and cognitive deficits. Moreover, we demonstrate that DKK1 elevated levels in blood circulation deregulates the immune responses in the brain and periphery. The effects of DKK1 widespread prolonged induction are sufficient to mediate persistent long-term damage. Our study indicates that DKK1 elevated levels play a determinant role in the pathobiology of cSVD associated with microinfarcts via deregulation of neurovascular and immune functions, highlighting its potential to develop novel prognostic tools and therapeutic interventions. 2. Material and Methods 2.1. Animal experiments Three to 5 months old transgenic mice [Gt(ROSA)26Sor tm1.1(rtTA,EGFP)Nagy Hprt tm1(tetO−Dkk1)Spdl Tg(TCF/Lef1-lacZ)34Efu/J; Stock N. 024746; The Jackson Laboratory] were used. The mice carry a knock-in allele containing Dkk1 gene under the control of tetO (tetO- Dkk1 ), inserted into the endogenous Hprt locus on X chromosome [ 29 ]. TetO- Dkk1 transgene was inserted via homologous recombination, allowing the integration of a single copy into a specific location in the genome and subsequent tissue-specific expression of the transgene [ 30 , 31 ]. The mice carry a Gt(ROSA)26Sor tm1.1(rtTA,EGFP)Nagy allele allowing a widespread expression of rtTA-EGFP as well as a Tg(TCF/Lef1-lacZ)34Efu reporter transgene. Widespread expression of Dkk1 gene was induced using doxycycline for 14 days (in diet; 200 mg/kg, Bio-Serv, NJ, USA), which triggers a conformational switch in rtTA, allowing its binding to tetO. The strain was kept on C57BL6/J background and C57BL6/J wildtype (WT) mice are used as controls. The following nomenclature was adopted to identify the activation state of DKK1 = DKK1/ON (transgenic mice with doxycycline), WT/DOX mice (WT mice with doxycycline), DKK1/ON→OFF (DKK1/ON mice upon doxycycline withdrawal) and WT/DOX→OFF (WT/DOX mice upon doxycycline withdrawal). Genotypes were confirmed by PCR as recommended by The Jackson Laboratory, using DNA isolated from ear punches and the following probes were used: Dkk1 : Common: 5’ – GGA AAA TGG CTG TGG TCA GA – 3’; Mutant forward: 5’- CCT ACC CTT CGC CTG AAG A – 3’; Wildtype forward: 5’- AGA AAA GTC TCT ACT GCC TCC AC – 3’, rTta: Wildtype reverse: 5’ – ATT CTG TGG GAA GTC TTG TCC – 3’; Wildtype forward: 5’ – CTT CCC TCG TGA TCT GCA AC – 3’; Mutant reverse: 5’ – GCT CTA ATG CGC TGT TAA TCA CT – 3’; Mutant forward: 5’ – GGT ACC CGC TCG AAA TTC A – 3’. All mice were housed under standard laboratory conditions and were provided with standard chow and water ad libitum. Animal experiments were conducted in accordance with the ethical policies and procedures approved by the committee of animal protection of Université Laval (Comité de Protection des Animaux de l'Université Laval (CPAUL)-3; Approval # 22-1136). Handling of animals adhered to the principles outlined in the Canadian Council on Animal Care (CCAC) guidelines. Animal studies were reported according to ARRIVE 2.0 guidelines. 2.2 cSVD associated with microinfarcts model Adult male mice between 25–30 g were subjected to micro-occlusions (MO) to generate cSVD associated with sporadic multifocal microinfarcts, as previously described [ 11 ]. Briefly, mice were anesthetized under 1.5% isoflurane in 1.5 l/minute (95% O2) and body temperature was maintained between 36 and 37°C using a feedback-controlled heating system (Harvard Apparatus, QC, Canada). A midline neck incision was performed to expose the left common carotid artery (CCA), the external carotid artery (ECA) and the pterygopalatine artery (PPA). The ECA and PPA were temporarily blocked using a microvascular clip under a surgical microscope (Leica Microsystems, ON, Canada) to ensure that all microspheres are directed towards the brain. Next, 2500 sterilized FITC-tagged microspheres of 20 µm (Polysciences Inc., PA, USA) suspended in 100 µL of PBS were slowly injected into the CCA using a 33G hypodermic needle (TSK Laboratory International, BC, Canada). Microspheres of 20 µm are used to occlude penetrating cerebral arterioles and capillaries [ 11 ]. After injection, the ECA and PPA were unblocked, the needle was gently removed from the CCA, and the bleeding was stopped by applying pressure using bioabsorbable Gelfoam (Pfizer, NY, USA). 2.3 Neurobehavioral analysis Animals underwent neurobehavioral tests prior to surgery (baseline) and at 14 and 30 days after cSVD onset. The open field (OF) test was used to assess anxiety-like behavior and general locomotor ability as previously described [ 32 ]. Mice were individually placed in an open arena (45 cm x 45 cm) and were allowed to move freely for 10 minutes. Animals’ movements were recorded, tracked, and analyzed using a video-tracking system (ANY-maze, Stoelting Co, IL, USA) in which the arena was divided into different zones namely, the center and corners. The parameters evaluated were the time spent in the center, the time spent in the corners, the total distance travelled and the total freezing time. An increase in the time spent in the center would indicate a disinhibitory behavior whereas a decrease shows an anxious state. A decrease in the distance could either mean a freezing state, a typical anxiety behavior, or motor impairment. The light/dark box test was used to assess anxiety-like behavior in rodents. The test is based on rodent’s innate aversion to brightly illuminated areas and on the spontaneous exploratory behavior of rodents in response to mild stressors, such as light [ 33 ]. The apparatus is composed of a compartment exposed to light and a dark comportment, separated by a wall with an open door. Each animal is first placed in the dark compartment and allowed to freely explore both compartments for 5 minutes. Latency to the first entry into the light compartment, time spent in the light compartment and the number of entries into the light compartment were recorded and analyzed. A reduced latency to enter the lightened compartment is interpreted as anxiety-like behavior in mice [ 34 ]. The novel object recognition (NOR) test was used to assess recognition memory, as previously described [ 18 , 35 ]. Briefly, during the acquisition phase, mice were individually allowed to explore two identical objects in an open arena (45 cm × 45 cm) for 10 minutes. Mice were returned to home cage for 1 hour and were placed back afterwards into the same arena, in the presence of one familiar object along with one novel object. Animals’ movements were objectively monitored and quantified with ANY-maze. Object exploration was defined as touching the object or directing the nose toward it at < 2 cm. We evaluated the time spent on both objects to assess mice performances. The arena and objects were cleaned with 70% alcohol between each mouse and trial. Animals whose exploration time was lower than 10 seconds were not used for analysis. A discrimination index (DI) ratio was calculated as follows = (time novel object − time familiar object)/(time novel object + time familiar object). DI ranges from + 1 to − 1; a positive ratio indicates a preference for the novel object, a negative ratio indicates more time spent with the familiar object, and a ratio of zero reflects no preference [ 36 ]. 2.4 Brain perfusion imaging and analysis Brain perfusion, which reflects the regional changes in cerebral blood flow (CBF), was investigated using Laser Speckle contrast imaging (LSCI) and laser Doppler flowmeter (LDF), as previously described [ 37 ]. Analysis was performed in the same animals to enable temporal assessment of brain perfusion at baseline, 5 minutes upon cSVD onset (immediate vascular responses) and on day 7 (delayed vascular responses). Briefly, mice were anesthetized using 1.5% isoflurane in 1.5 l/min (95% O2). Mice head was shaved, 100 µL of lidocaine/bupivacaine solution was applied on the incision site and the ears (50 µL/ear). Mice were placed on a stereotaxic frame (RWD Life Science Inc., CA, USA), the skull was exposed by removing the skin using fine-tip forceps, and brain perfusion was visualized using 2D laser Speckle blood flow imager (OMEGAZONE OZ-3, Omegawave Inc., Japan). The OZ-3 system is equipped with a visible and near infra-red (NIR) CCD camera, continuously displaying real images while comparing color differences between real and blood flow images. Brain perfusion in each hemisphere was calculated by averaging 20 consecutive raw Speckle images in each ROI using a built-in software. The ratio of blood perfusion in the ipsilateral over contralateral hemisphere was calculated. Brain perfusion was further analyzed using LDF (OMEGAFLOW FLO-N1; Omegawave Inc.). A non-contact fiber optic probe was positioned at 1 cm distance from the top of the skull covering the ipsilateral and contralateral hemisphere. The mean values of blood flow (i.e. brain perfusion), velocity, and mass were recorded for 20 seconds for each ROI and processed using a built-in software. 2.5 Brain tissue processing Mice were euthanized 14 days and 1 month after cSVD onset. Briefly, mice were deeply anesthetized with a solution containing ketamine (90 mg/ml) and xylazine (10 mg/ml) then euthanized via transcardiac perfusion with PBS followed by 1% paraformaldehyde (PFA). Brains were extracted and post-fixed in 4% PFA for 24 hours followed by 4% PFA with 20% sucrose for 24 hours. Brains were cut into 25 µm coronal sections on microtome (Leica Biosystems, ON, Canada), and sections were collected in 12 well-plates containing an antifreeze solution (30% glycerol, 30% ethylene glycol in 0.9% sodium chloride (NaCl), phosphate buffer (PB)) and kept at -20°C for further use. 2.6 Immunofluorescence analysis Free-floating brain sections were first rinsed 3x in KPBS for 10 minutes and then incubated for 1 hour in a permeabilization/blocking solution containing 4% normal donkey serum (NDS) (Sigma-Aldrich, D9663), 1% bovine serum albumin (BSA) (Wisent Inc., 800-095-CG), and 1% Triton X-100 (Fisher scientific, BP151-100) in KPBS. Brain sections were then incubated overnight at room temperature with different primary antibodies diluted in the same solution. The following primary antibodies were used; chicken anti-NeuN (1:1000, MilliporeSigma, ABN91), rabbit anti-doublecortin (DCX) (1:1000, Abcam, ab207175), rabbit anti-ionized calcium binding adaptor molecule (IBA-1) (1:500, WAKO, 019-19741), rat anti-CD45 (1:500, BD Biosciences, 553076). The next day, brain sections were rinsed 3x in KPBS for 10 minutes and incubated for 2 hours at room temperature with the adequate secondary antibodies at a 1:700 dilution in KPBS; Cy3 donkey anti-chicken (Millipore Sigma, AP194C), Alexa Fluor 647 donkey anti-rabbit (Invitrogen, A31574), Alexa Fluor 647 donkey anti-rat (Jackson ImmunoResearch Laboratories, 712-605-153). Brain sections were then rinsed 3x with KPBS and incubated with 4′,6-diamidino-2-phenylindole (DAPI) (1:10 000) for 5 minutes. Brain sections were rinsed with KBPS, mounted onto Superfrost® micro-slides, and cover-slipped with Fluoromount-G® anti-fade medium (Electron Microscopy Science, 17984-25). Epifluorescence images were acquired using Axio Observer microscope equipped with optical sectioning (Apotome.2) and Axiocam 503 monochrome camera and processed using with ZEN Imaging Software (Carl Zeiss Canada, ON, Canada). NeuN + area in the hippocampus, DCX intensity and projection length, IBA-1 area and intensity and CD45 intensity were measured with FIJI software. 2.7 Immunohistochemical analysis Immunohistochemical analysis was used to assess extravasation of blood-borne immunoglobulin G (IgG), an endogenous marker of BBB breakdown, as previously described [ 38 ]. Brain sections were rinsed with KPBS (Sigma-Aldrich) and then incubated for 20 minutes in a permeabilization/blocking solution containing 4% normal goat serum (NGS; Sigma-Aldrich, GG9023), 1% BSA, and 0.4% Triton X-100 in KPBS. Brain sections were incubated overnight at 4°C with biotinylated goat anti-mouse IgG antibody (1/1000; BA-9200; Vector laboratories) in the same permeabilization/blocking solution. The next day, brain sections were rinsed 3x with KPBS and then incubated for 1 hour at room temperature with Avidin Biotin Peroxidase Complex (ABC) (Vectastain Elite Kit Standard). Brain sections were rinsed 3x with KPBS before incubation with 3,3’-diaminobenzidine tetrahydrochloride (DAB, Sigma-Aldrich) for 10 minutes, washed 3x with KPBS. Brain sections were mounted onto slides, dried, dehydrated via immersion in an increased concentration of ethanol (EthO) solution (50%, 70%, 75%, 95% and 100%), and finally immersed in Xylene solution 3x for 3 minutes and cover-slipped using DPX mounting solution (Electron Microscopy Sciences). Mounted sections were digitized and the intensity of DAB signal (optical density) in the ipsilateral and contralateral (i.e. background) cortex and hippocampus was analyzed using FIJI software. Ratio of ipsilateral/contralateral intensity was computed and values > 1 indicates an increased IgG intensity (i.e. infiltration) in the ipsilateral brain structures. 2.8 Enzyme-linked immunosorbent assay (ELISA) Mouse DKK1 Quantikine ELISA Kit (R&D Systems, MKK100) was used to quantify DKK1 concentration in the serum at 14 and 30 days after cSVD. Briefly, blood samples were collected from the submandibular vein and left at room temperature for 2 hours to allow clotting. The samples were then centrifuged at 2000 g for 20 minutes, after which the serum was separated, aliquoted, and stored at − 20°C until analysis. All samples were assayed in duplicates. Serum samples of DKK1/ON mice were diluted 1:50 and serum samples of WT/DOX, WT/DOX→OFF and DKK1/ON→OFF were diluted 1:10. The optical density was determined using a microplate reader (SpectraMax i3, Molecular Devices, CA, USA) at 450 nm with wavelength correction at 540 nm and analyzed using SOFTmax Pro 6.4.0.1 software (Molecular Devices). All experiments were performed according to the manufacturer's instructions (R&D Systems, MKK100). 2.9 Cytokine/chemokine array The cytokine and chemokine concentrations in the serum of WT/DOX→OFF and DKK1/ON→OFF at 30 days after cSVD were analyzed at Eve Technologies (Calgary, Canada) using the Mouse Cytokine Proinflammatory Focused 10-Plex Discovery Assay Array (Eve Technologies, MDF10). 2.10 Flow cytometry analysis Blood samples were collected from the submandibular vein into ethylene-diamine-tetra-acetic acid (EDTA) coated vials (Sarstedt, Montréal, QC, Canada). Analysis of the dynamics of monocytes was performed as previously described [ 39 ]. Briefly, 60 µL of total blood was incubated at room temperature for 5 minutes with 1 mL of ACK (ammonium, chloride, potassium) lysing buffer to get rid of red blood cells. Samples were then washed with 3 mL of PBS and centrifuge at 400 g for 8 minutes at 4°C. The supernatant was removed, and cells were resuspended with blockage solution (1 µL CD16/CD32 antibody (BD bioscience) diluted in 100 µL of PBS per tube) and incubated for 10 minutes on ice. Then, samples were incubated for 30 minutes in the dark on ice with 100 µL of the following antibody mix: cluster of differentiation (CD)-45 BV605 (1:400, Biolegend, 103139), CD11b Pe-Cy7 (1:100, EBioscience, 25–0112), CD115 APC (1:75, EBioscience, 17-1152), Ly6C V450 (1:100, BD Bioscience, 560594), Ly6G Pe (1:200, BD bioscience, 551461) and live/dead AF 780 (1:400, BD bioscience, 565388). After incubation, the samples were washed with 3 mL PBS, centrifuged for 8 minutes at 400 g, resuspended in 250 µL of PBS, and complemented with 50 µL of 123count eBeads™ Counting Beads (Invitrogen, 01-1234-42). Samples were processed using a LSR II flow cytometer, and data was acquired using BD FACS Diva software (Version 6.1.2, BD Bioscience), and analyzed using FlowJo software v10 (Tree Star; Ashland, OR, USA). 2.11 Statistical analysis Data are presented as boxplot with min/max whiskers or mean ± standard deviation (SD). Data distribution normality was assessed with the Shapiro Wilk test. If data followed normal distribution, unpaired two-tailed t-test was used for comparison between two groups, and one-way or two-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test were used for multiple comparison. If data did not follow normal distribution, Mann-Whitney test was used for comparison between two groups and Kruskal-Wallis test followed by Dunn’s test was used for multiple comparison. P < 0.05 was considered statistically significant. Statistical analyses were carried out using GraphPad Prism (version 10, GraphPad Software, San Diego, CA, USA). 3. Results 3.1. cSVD is associated with a prolonged increase in DKK1 expression in blood circulation Previous reports have indicated that circulating DKK1 levels are elevated in various vascular pathologies, including stroke [ 20 – 23 ], as well as vascular risk factors [ 16 , 24 – 28 ]. Herein, we aimed first to investigate the temporal regulation of DKK1 expression in blood circulation upon cSVD associated with microinfarcts. Our ELISA analysis showed that DKK1 levels in serum were rapidly increased in C57BL6/J mice upon cSVD at 24 hours [+ 6.26%] and remained elevated until day 14 to subsequently return to basal levels at day 30 after onset [-17.86%] ( Fig. 1 a ) . This suggests that cSVD onset is associated with a prolonged release of DKK1 in blood circulation that persists over time before normalization. To further characterize the role of DKK1, C57BL6/J mice (WT/DOX) and inducible DKK1 transgenic mice (DKK1/ON) on doxycycline (200 mg/kg; 14 days) were subjected to cSVD. In one cohort of mice, doxycycline was kept for 14 days after onset to maintain DKK1 induction, while in another cohort, doxycycline was withdrawn between day 14 and day 30 after onset, to normalize DKK1 expression (WT/DOX→OFF, DKK1/ON→OFF) ( Fig. 1 b ) . Using ELISA, we showed that DKK1 circulating levels at day 14 after cSVD were significantly induced in DKK1/ON mice (127 375.4 ± 128 216.2 pg/mL) compared to WT/DOX mice (20 118.6 ± 3315.2 pg/mL) [P = 0.0014] ( Fig. 1 c ) . Upon doxycycline withdrawal, DKK1 circulating levels at day 30 were similar in DKK1/ON→OFF mice (14 162.2 ± 4923.4 pg/mL) and WT/DOX→OFF mice (16 135.1 ± 5431.2 pg/mL) [P = 0.3653] ( Fig. 1 d ) . Moreover, DKK1 elevated levels significantly exacerbated weight loss in DKK1/ON mice compared to WT/DOX mice at day 3 [P = 0.0452], day 4 [P = 0.0111] and day 6 [P = 0.0325] after cSVD ( Fig. 1 e ) . Upon normalization of DKK1 levels, DKK1/ON→OFF mice still had greater weight loss at day 30 after cSVD to WT/DOX→OFF [P = 0.0058] ( Fig. 1 e ) . Our findings indicate that cSVD is associated with prolonged transient increase in DKK1 levels in blood circulation, correlating with an exacerbated alteration of the overall physical state of mice. 3.2. Excessive DKK1 circulating levels exacerbate structural and functional vascular integrity Deregulation of neurovascular functions, associated with alteration of the vascular permeability and cerebral microcirculation, play a central role in the pathobiology of cSVD [ 28 , 40 , 41 ]. We first assessed the impact of DKK1 circulating elevated levels on vascular permeability via analysis blood-borne IgG extravasation into the brain 14 days after cSVD (Fig. 2 a). IgG infiltration was higher in the ipsilateral hemisphere of DKK1/ON mice compared to WT/DOX mice [P = 0.0541] ( Fig. 2 b ) , which was less prominent in the hippocampus [P = 0.1520] ( Fig. 2 c ) , probably due to the sporadic micro-occlusions that generate spatially heterogenous microinfarcts in restricted brain regions. Next, we investigated the consequences of DKK1 induction on temporal brain perfusion upon cSVD using laser Speckle contrast imaging (LSCI) and non-contact laser Doppler flowmetry (LDF). LSCI was performed at baseline prior to cSVD, 5 minutes upon micro-occlusions (immediate impact of vascular reactivity) and 7 days (delayed vascular reactivity) after cSVD onset in WT/DOX and DKK1/ON mice ( Fig. 2 d ) . LSCI analysis revealed that DKK1 induction potently reduced brain perfusion immediately (5 minutes) upon cSVD onset in the ipsilateral hemisphere [P = 0.0066] ( Fig. 2 e ) but not in the contralateral hemisphere [P = 0.1466] ( Fig. 2 f ) of DKK1/ON mice compared to WT/DOX mice. Brain perfusion was completely recovered in the ipsilateral [P = 0.1872] and contralateral [P = 0.6848] hemispheres of WT/DOX and DKK1/ON mice at day 7 after cSVD ( Fig. 2 e, f ) . Furthermore, our analysis showed that DKK1 elevated levels are associated with an overall greater attenuation of brain perfusion immediately (5 minutes) upon cSVD onset in the ipsilateral hemisphere relatively to the contralateral hemisphere of DKK1/ON mice compared to WT/DOX mice [P = 0.0263] ( Fig. 2 g ) . This suggests that DKK1 elevated levels impair acute vascular reactivity in response to micro-occlusions, leading to an exacerbated reduction of cerebral microcirculation. These results were corroborated with LDF analysis that has outlined a significant reduction in blood flow [P = 0.0267] ( Fig. 2 h ) as well as velocity [P = 0.0362] ( Fig. 2 i ) immediately (5 minutes) after cSVD in the ipsilateral hemisphere relatively to the contralateral hemisphere of DKK1/ON mice compared to WT/DOX mice. Our findings show that DKK1 elevated levels exacerbate vascular structural integrity increasing permeability and alter acute vascular reactivity, thereby impairing brain perfusion after cSVD. 3.3. DKK1 induction aggravates neuronal loss and impairs neurogenesis in the hippocampus The hippocampus, which plays a major role in various cognitive functions such as anxiety and memory, is highly vulnerable to ischemic injury [ 41 ]. It has been previously shown that cSVD mediates neurodegenerative responses in the hippocampus [ 42 ]. Herein, we aimed to evaluate the impact of elevated circulating DKK1 levels on neuronal damage after cSVD particularly in the hippocampus. NeuN immunolabelling in the hippocampus at 14 and 30 days after cSVD was used to assess neuronal loss ( Fig. 3 a ) . Our analysis at 14 days after cSVD indicated that WT/DOX mice displayed a significant reduction of NeuN + area in the CA of the ipsilateral hippocampus when compared to the area of matched intact CA of the contralateral hemisphere [P = 0.0043] ( Fig. 3 b ) . However, the reduction was more important in DKK1/ON mice [P = 0.0002], indicating DKK1 elevated levels led to greater neuronal loss in the CA region ( Fig. 3 c ) . Evaluation of neuronal loss at 30 days showed that WT/DOX→OFF mice still exhibited an important reduction in NeuN + area in the CA of the ipsilateral hippocampus in comparison to the matched intact CA of the contralateral hemisphere [P = 0.0006] ( Fig. 3 d ) , which was less prominent in DKK1/ON→OFF mice [P = 0.0433] (Fig. 3 e). This suggests that normalization of DKK1 levels upon doxycycline withdrawal prevents further neuronal loss after cSVD, and that the extent of neuronal loss is solely attributed to micro-occlusions. Next, we assessed the neurogenic response in the dentate gyrus (DG) by evaluating the turnover of neuronal progenitor cells (NPCs) using doublecortin (DCX) immunolabelling (Fig. 3 f). We first investigated basal neurogenesis in the contralateral DG, and observed that DCX mean intensity, indicative of the overall density of NPCs, was similar between WT/DOX and DKK1/ON mice at day 14 after cSVD (Fig. 3 g). However, DCX mean intensity was significantly reduced in the contralateral DG of DKK1/ON→OFF mice compared to WT/DOX→OFF mice [P = 0.0003] at day 30 after cSVD (Fig. 3 g). On the other hand, DCX mean intensity was increased in WT/DOX mice compared to DKK1/ON mice in the ipsilateral DG at day 14 after cSVD [P = 0.0128], which was maintained upon normalization of DKK1 expression in WT/DOX→OFF mice compared to DKK1/ON→OFF mice at day 30 days [P = 0.0197] (Fig. 3 h). Next, we assessed the length of DCX + cell projections, indicative of overall dendritic integrity [ 43 ]. In the contralateral DG, the mean length of DCX + cell projections was greater in DKK1/ON mice compared to WT/DOX mice at day 14 after cSVD [P = 0.0401] but was significantly reduced in DKK1/ON→OFF mice compared to WT/DOX→OFF mice at day 30 [P < 0.0001] (Fig. 3 i). In the ipsilateral DG, the mean length of DCX + cell projections remained unchanged at day 14 after cSVD between WT/DOX and DKK1/ON mice but was significantly reduced in DKK1/ON→OFF mice compared to WT/DOX→OFF at day 30 after cSVD [P < 0.0001] (Fig. 3 j). Our results suggest that DKK1 excessive release at cSVD onset mediates a progressive impairment of neurogenesis despite delayed normalization of expression levels. 3.4. Excessive DKK1 expression promotes hyperactive behavior independently upon cSVD cSVD contributes to cognitive decline and neuropsychiatric symptoms [ 41 , 44 ]. Furthermore, various reports have indicated that DKK1 is implicated in the pathological processes underlying stress-related disorders [ 45 , 46 ]. Hence, we investigated the impact of elevated DKK1 circulating levels after cSVD on anxiety-like behaviors and general ambulatory ability using the open field test (Fig. 4 a). For time spent in the center, two-way analysis of variance (ANOVA) revealed no interaction between DKK1 induction state (WT mice and inducible DKK1 transgenic mice with or without DOX) and time associated with cSVD onset (baseline, 14 days, 30 days) [P = 0.6233] (Fig. 4 b). However, the main effect of time associated with cSVD onset was significant [P = 0.0009] while the main effect of DKK1 induction state was not [P = 0.2275], indicating the time spent in the center was affected essentially by cSVD, regardless of DKK1 levels (Fig. 4 b). For time spent in the corners, two-way ANOVA revealed no interaction between DKK1 induction state and time associated with cSVD onset [P = 0.5276] (Fig. 4 c). Similarly, the main effect of time associated with cSVD onset was significant [P < 0.0001] while the main effect of DKK1 induction state was not [P = 0.9525], suggesting that time spent in corners was affected mainly by cSVD, independently upon DKK1 levels (Fig. 4 c). Although there was still no interaction for the total distance travelled [P = 0.1895], the main effect of time associated with cSVD onset [P < 0.0001] as well DKK1 induction state [P = 0.004] were both significant, indicating that the total distance travelled in the open field was affected by cSVD as well as DKK1 elevated levels (Fig. 4 d). Tukey’s post-hoc test revealed that DKK1 induction increased overall mobility, translated by a more important distance travelled at baseline [P = 0.0015] and 14 days after cSVD [P = 0.0203] in inducible DKK1 transgenic mice compared to WT mice with DOX. Notably, there was no difference 30 days after cSVD between WT and inducible DKK1 mice without DOX [P = 0.4393] (Fig. 4 d), suggesting that mobility was restored upon normalization of DKK1 levels. For the freezing time, two-way ANOVA showed no interaction between the two factors [P = 0.3897], but the main effect of time associated with cSVD [P < 0.0001] and DKK1 induction state [P = 0.0027] were significant (Fig. 4 e). Tukey’s post-hoc test showed that inducible DKK1 transgenic mice with DOX exhibited reduced freezing time at day 14 [P = 0.0194] and day 30 after cSVD [P = 0.0215] (Fig. 4 e). This indicates that while DKK1 induction did not affect the time spent in the different zones of the open field, it increased the total distance travelled and reduced the freezing time, outlining the emergence of hyperactive behavior. We further explored the anxiety-like behavior using the light and dark box test (Fig. 4 f). Two-way ANOVA of the latency of first entry in the light box showed no interaction between DKK1 induction state and time associated with cSVD onset [P = 0.7658], in absence of independent main effects of DKK1 induction state [P = 0.3479] or time associated with cSVD onset [P = 0.3954] (Fig. 4 g). Similar results were obtained for the number of entries in the light box with no significant interaction between the two factors [P = 0.2613], in absence of independent main effects of DKK1 induction state [P = 0.4436] or time associated with cSVD onset [P = 0.6055] (Fig. 4 h). There was no interaction between DKK1 induction state and time associated with cSVD onset on the total exploration time in the light box [P = 0.9891], with no independent main effects of DKK1 induction state [P = 0.1731] or time associated with cSVD onset [P = 0.2614] (Fig. 4 i). These results suggest neither time after cSVD nor DKK1 elevated levels significantly influenced anxiety-like behaviors in mice in the light and dark box test. Next, we used novel object recognition (NOR) test to assess recognition memory (Fig. 4 j). Two-way ANOVA revealed no interaction between DKK1 induction state and time associated with cSVD onset for the total exploration time of both objects [P = 0.7953], but the main effect of time associated with cSVD onset was significant [P = 0.0249], indicating that the innate instinct to explore objects varied over time after cSVD, independently of the main effect of DKK1 induction state [P = 0.2637] (Fig. 4 k). However, analysis of the discrimination index showed no interaction between DKK1 induction state and time associated with cSVD onset [P = 0.6648], with no independent main effects of time associated with cSVD onset [P = 0.3578] and DKK1 induction state [P = 0.9647], indicating that recognition memory didn’t vary over time upon cSVD nor with DKK1 elevated levels (Fig. 4 l). Our findings suggest that DKK1 elevated levels promote hyperactive state independently upon cSVD. 3.5. cSVD-mediated neuroinflammation is minimally affected by DKK1 induction Neuroinflammation underlying ischemic lesions could be detrimental, leading to injury progression when imbalanced, yet also contributes to tissue repair [ 47 ]. Microglia, brain’s resident immune cell, and infiltrating myeloid cells are rapidly activated and recruited to the lesion sites upon cSVD associated with microinfarctions, to partake in the inflammatory responses [ 11 , 48 ]. As DKK1 is a potent immunomodulator [ 49 ], we aimed to evaluate the impact of its elevated levels on the neuroinflammatory responses. Immunofluorescence analysis showed that the intensity of IBA-1 ( Fig. 5 a ) , expressed in microglia and monocyte-derived macrophages (MDMs), slightly increased in the ipsilateral hemisphere of DKK1/ON mice compared to WT/DOX mice at day 14 after cSVD [P = 0.0939] (Fig. 5 b). At day 30 after cSVD, following normalization of DKK1 levels, IBA-1 intensity remained unchanged between both groups [P = 0.3148] (Fig. 5 b). Similarly, in the hippocampus, IBA-1 intensity remained unchanged between both groups at 14 days [P = 0.1206] and slightly decreased in DKK1/ON→OFF mice upon normalization of DKK1 levels at 30 days after cSVD [P = 0.0878] (Fig. 5 c). Next, we assessed the extent of the area covered by reactive IBA-1 + cells, and we observed a slight increase in the ipsilateral hemisphere of DKK1/ON mice compared to WT/DOX mice at day 14 after cSVD [P = 0.1260] (Fig. 5 d). No difference in reactive IBA-1 + cell coverage was observed at day 30 after cSVD upon normalization of DKK1 levels [P = 0.2596] (Fig. 5 d). Similarly, reactive IBA-1 + coverage in the hippocampus remained unchanged between both groups at day 14 [P = 0.2610] and day 30 [P = 0.1535] after cSVD (Fig. 5 e). Our findings indicate that elevated DKK1 levels slightly enhanced microglial reactivity after cSVD, but this effect was completely resolved after normalization of DKK1 expression. Next, we evaluated the overall infiltration of immune cells by immunolabeling CD45, which is also expressed in MDMs ( Fig. 5 f ) . Our analysis revealed that CD45 intensity in the ipsilateral hemisphere was similar between both groups at day 14 after cSVD [P = 0.2810] (Fig. 5 g). Analysis of the mean CD45 fluorescence signal revealed no significant difference between WT/DOX mice (181.81±119.99) and DKK1/ON mice (430.93±497.07), but we could appreciate a slight overall increase in DKK1/ON mice at day 14 after cSVD [P = 0.2810] (Fig. 5 h). At day 30 after cSVD, CD45 intensity remained unchanged in WT/DOX→OFF mice compared to DKK1/ON→OFF mice [P = 0.1331] (Fig. 5 i). Analysis of the mean CD45 fluorescence signal in the ipsilateral hemisphere outlined an increase in WT/DOX→OFF mice (46.19±43.91) compared to DKK1/ON→OFF mice (1.07±1.09) [P = 0.0001] (Fig. 5 j). This suggests that while the peak of neuroinflammation at day 14 after cSVD is not affected by DKK1, its resolution is accelerated upon normalization of its expression levels. Our findings indicate that neuroinflammatory responses after cSVD are not significantly affected by DKK1 expression levels. 3.6. DKK1 elevated levels maintain a pro-inflammatory phenotype in monocytes upon cSVD Circulating monocytes play an important role in the pathobiology of cSVD associated with microinfarcts [ 42 , 50 ]. Murine monocytes can be classified based on Ly6C expression and comprise pro-inflammatory Ly6C high , patrolling Ly6C low and intermediate Ly6C inter subsets [ 51 ]. Our group has previously demonstrated that monocyte subsets are differentially regulated upon cSVD [ 41 ]. Herein, we used flow cytometry analysis to elucidate the impact of DKK1 elevated circulating levels on the dynamics of different monocyte subsets at baseline, 14 and 30 days after cSVD (Fig. 6 a). Prior to cSVD, frequencies in total leukocytes (CD45 + ) [P = 0.9430] and total monocytes (CD11b + CD115 + ) [P = 0.7271], as well as Ly6C low [P = 0.6821], Ly6C inter [P = 0.7056] and Ly6C high [P = 0.6154] subsets showed no differences between WT/DOX mice and DKK1/ON mice, indicating that DKK1 induction didn’t affect basal monocyte distribution (Fig. 6 b-f). Furthermore, frequencies of total leukocytes [P = 0.7396] and total monocytes [P = 0.1484] between the two groups remained unchanged at day 14 after cSVD (Fig. 6 g, h). Although no significant differences in the frequencies of monocyte subsets at day 14 after cSVD were reported, we observed a slight decrease in the frequency of Ly6C low in DKK1/ON mice compared to WT/DOX mice [P = 0.1018] (Fig. 6 i), no difference in the frequency of Ly6C inter [P = 0.7833] (Fig. 6 j), and a slight increase in the frequency of Ly6C high [P = 0.1180] (Fig. 6 k) subset in DKK1/ON mice compared to WT/DOX mice. This suggests that circulating monocytes are acquiring a pro-inflammatory phenotype after cSVD when DKK1 circulating levels are elevated. At day 30 after cSVD following normalization of DKK1 levels, no difference in the frequencies of total leukocytes [P = 0.6197] and total monocytes [P = 0.3638] were noted between both groups (Fig. 6 l, m). However, frequency of Ly6C low monocytes significantly increased in WT/DOX→OFF mice compared to DKK1/ON→OFF mice [P = 0.0003] (Fig. 6 n ) . Frequency of Ly6C inter monocytes significantly decreased in DKK1/ON→OFF mice compared to WT/DOX→OFF mice [P = 0.0003] (Fig. 6 o). Moreover, frequency of Ly6C high monocytes was strongly reduced in WT/DOX→OFF mice compared to DKK1/ON→OFF mice [P < 0.0001] (Fig. 6 p). This indicates that monocytes in WT mice respond to cSVD by increasing the frequency of patrolling Ly6C low subset over time (Fig. 6 q) to partake in neurovascular repair [ 42 ]. However, DKK1 elevated levels mediate profound changes in the response of monocytes over time, by maintaining an elevated frequency of pro-inflammatory Ly6C high subset involved in cSVD progression [ 52 ] (Fig. 6 q). Our findings show that DKK1 elevated levels in blood circulation promote an acute-like long lasting pro-inflammatory polarization of monocytes after cSVD. 3.7. Circulating DKK1 differentially regulates anti- and pro-inflammatory cytokines in serum To further explore the long-term effects of DKK1 induction on systemic inflammatory responses, we quantified the concentration of key cytokines in the serum of WT/DOX→OFF and DKK1/ON→OFF mice 30 days after cSVD, following normalization of DKK1 levels. Granulocyte macrophage colony stimulating factor (GM-CSF), a multipotent cytokine that can stimulate proliferation of bone marrow derived macrophages and granulocytes [ 53 ], was detected in 50% of WT/DOX→OFF mice at a mean concentration of 6.33 pg/mL [5.66–7.00 pg/mL], while it was undetectable in all DKK1/ON→OFF mice ( Table 1 ). Anti-inflammatory cytokine interleukin 10 (IL-10) was detected in 50% of both groups with a mean concentration of 2.93 pg/mL [0.36–5.50 pg/mL] in WT/DOX→OFF mice and 1.93 pg/mL [1.74–2.11 pg/mL] in DKK1/ON→OFF mice, suggesting a slight decrease of IL-10 in the DKK1 transgenic mice ( Table 1 ). Finally, interferon-γ (INF-γ), a pro-inflammatory mediator, was detected in 25% of WT/DOX→OFF mice at a concentration of 1.63 pg/mL and was detected in 75% of DKK1/ON→OFF mice with a mean concentration of 1 pg/mL [0.38–2.14 pg/mL] ( Table 1 ). Our findings suggest that DKK1 high levels in blood circulation at cSVD onset mediate prolonged changes associated with the release of pro-inflammatory cytokines. 4. Discussion A better understanding of the mechanisms modulating the pathobiology of cSVD associated with microinfarcts would provide novel insights into the development of effective therapeutic approaches. The current prevention and treatment strategies rely essentially on managing vascular risk factors associated with cSVD, such as hypertension, diabetes, hypercholesterolemia and smoking, through lifestyle modifications and pharmacological interventions [ 54 , 55 ]. DKK1 is an antagonist of the canonical Wnt pathway which plays a key role in maintaining neurovascular integrity and regulating immune functions [ 14 ]. DKK1 circulating levels are elevated in different pathological conditions, including vascular, immune and metabolic disorders [ 26 , 27 , 56 ]. This suggests that DKK1 represents a major factor potentially implicated in aggravating the progression of cSVD, outlining its potential as a prognostic and therapeutic target. Herein, we show that DKK1 circulating levels potently increase in the days following cSVD, gradually decreasing over time. DKK1 circulating levels have been reported to be elevated in stable cerebrovascular diseases [ 20 ]. Nonetheless, our findings suggest that cSVD associated with microinfarcts triggers DKK1 de novo release in the blood circulation. As DKK1 elevated levels is commonly reported in various risk factors associated with cSVD, we further elucidated the impact of its conditional induction in modulating the pathobiology of cSVD. This was achieved using innovative tools to mediate DKK1 widespread induction while preserving its endogenous tissue distribution. Our analysis indicates that DKK1 induction in transgenic mice results in greater short-term and long-term weight loss after cSVD. Various reports indicate that weight loss is linked to poor functional outcomes and mortality risk after ischemic stroke and positively correlates with infarct volume and severity, suggesting it could be detrimental as well in the context of cSVD [ 57 , 58 ]. DKK1 widespread expression also tends to exacerbate vascular permeability in the ipsilateral hemisphere, which represents a critical pathological factor in cSVD pathogenesis etiology and clinical progression [ 59 , 60 ]. Additionally, we reveal that DKK1 elevated levels are associated with a severe interruption of CBF upon occlusion of the penetrating arterioles. This suggests that elevated DKK1 levels at cSVD onset alter acute vascular reactivity and hinder immediate brain reperfusion, thereby compromising acute neurovascular responses, which underly exacerbation of ischemic injury severity [ 61 ]. The hippocampus is particularly vulnerable to hypoperfusion mediated by CBF impairments associated with cerebrovascular pathologies, including dementia [ 62 ]. Our group has previously shown that cSVD associated with microinfarcts implicates important neuronal loss in the hippocampus [ 41 , 42 ]. Herein, we report that DKK1 elevated levels exacerbate neuronal loss specifically in the CA region. Interestingly, normalization of DKK1 widespread expression after cSVD via withdrawal of doxycycline reduces progressive neuronal loss. This suggests that strategies aiming to block DKK1 action could promote neuroprotection effects in cSVD. It is in line with previous reports indicating that attenuation of DKK1 expression protects neurons in the hippocampus against ischemic insults [ 25 ]. Brain injuries activate neurogenic niches, including DG in the hippocampus, translated by the proliferation, migration, and differentiation of neural stem cells, translating an attempt to replace the lost neurons [ 63 , 64 ]. Evidence is indicating that microinfarcts associated with VaD implicate activation of neurogenic responses [ 65 , 66 ]. Herein, we uncover that DKK1 induction impairs neurogenesis under normal conditions. Notably, DCX + neuroblasts in the intact DG upon DKK1 induction exhibit atrophic dendrites. Canonical Wnt pathway regulates dendrite morphogenesis, and its inhibition reduces dendritic length, which could lead to weaker network connectivity [ 43 , 67 ]. Furthermore, activation of canonical Wnt signaling mediated via reduction of DKK1 expression has been demonstrated to enhance neurogenesis in the hippocampus [ 43 ]. Our results indicate that DKK1-induced reduction of neurogenesis, before and after cSVD, impairs endogenous neurorestorative mechanisms. cSVD is associated with various clinical manifestations depending upon the affected brain region [ 6 , 7 ]. We show that anxiety-like behaviors in mice are increased upon cSVD and that DKK1 elevated levels are associated with the emergence of a hyperactive behavior. Hyperactivity or increased spontaneous activity in rodents, presumably translating an agitated state, are often observed in VaD as well as at the early stages of AD [ 68 , 69 ]. Neuroinflammation plays an important role in the pathogenesis of cSVD [ 70 ]. DKK1 acts as an immunomodulator under various pathological conditions [ 70 ]. Herein, we aimed to investigate the impact of DKK1 elevated levels on immune responses in cSVD associated with microinfarcts. Microglial cell activation plays a multifaceted role in brain injuries associated with infarctions by either contributing to tissue repair via removal of debris and release of trophic factors, or exacerbating damage via excessive release of pro-inflammatory mediators [ 11 , 71 , 72 ]. In our study, we show that DKK1 elevated levels enhance microglial reactivity after cSVD, which is attenuated over time upon normalization of its expression levels. Infiltrating myeloid cells represent as well an important component of neuroinflammation after injury [ 51 ]. Our analysis suggests that peripheral immune cell infiltration slightly increases with DKK1 elevated levels, an effect that is reversed upon normalization of its expression levels. This suggest the DKK1 levels may contribute to the establishment of an inflammatory microenvironment that impedes repair. In addition to resident microglia, ischemic insults trigger the recruitment of monocytes that differentiate into MDMs [ 41 , 73 ]. Our group previously showed that monocyte subsets are differentially modulated after cSVD, affecting injury progression [ 41 ]. Herein, we reveal that circulating Ly6C high inflammatory monocytes progressively transition into Ly6C low monocytes, which play a critical role in preserving neurovascular functions upon cSVD [ 42 ]. However, DKK1 elevated levels promote a persisting pro-inflammatory phenotype in circulating monocytes after cSVD despite normalization of its expression levels. Interestingly, Ly6C high inflammatory monocytes are characterized by an increased capacity of cytokine production and are linked to cSVD progression [ 52 ]. This suggests that DKK1 elevated levels promote the accumulation of pro-inflammatory monocytes in blood circulation, possibly contributing to exacerbation of systemic inflammation and subsequently injury progression upon cSVD. Different studies outlined an association between circulating inflammatory cytokines with cSVD onset and progression [ 74 ]. Although GM-CSF is mainly recognized as a pro-inflammatory cytokine, evidence suggests it could act as an anti-inflammatory cytokine depending on the context [ 75 ]. Herein, we show that GM-CSF is undetectable in inducible DKK1 transgenic mice after cSVD upon DKK1 normalization, correlating with present systemic inflammation as indicated by the increased frequency of pro-inflammatory monocytes. Nonetheless, GM-CSF is detected in 50% of WT mice in which an increased frequency of Ly6C low monocyte is reported. This outlines a contribution to resolution of inflammation under normal conditions after cSVD. IL-10 is detectable in 50% of both groups, but at slightly lower levels in inducible DKK1 transgenic mice upon DKK1 normalization. IL-10 is endogenously produced by immune cells to limit the inflammatory reaction induced by ischemic insults and its administration has been shown to provide neuroprotection in experimental stroke [ 76 ]. INF-γ is a pro-inflammatory mediator that contributes to inflammatory and thrombogenic responses in ischemic stroke [ 77 ]. We detected INF-γ in 75% of inducible DKK1 transgenic mice and in only 25% of WT mice. Herein, we uncover that DKK1 elevated levels at cSVD onset is sufficient to mediate profound changes in the systemic inflammatory responses that persist over time despite normalization of its circulating levels, an aspect that should be taken into consideration. Our study provides new insights into the role of DKK1 in cSVD pathobiology via deregulation of immune-neurovascular functions. Our findings suggest that DKK1 circulating levels would provide valuable information about brain injury progression associated with cSVD and outline its potential as a promising target for the development of clinically relevant disease-modifying therapies. Declarations 5. Acknowledgements We would like to thank Natija Aldib for helping in the LSCI experiments. 6. Author contributions Authors contributed as follows; Esther Trudel: Experimentation, data interpretation, figure preparation, drafting and editing. Anne-Sophie Allain: Experimentation, and editing. Ayman ElAli; Conceptualization, data interpretation, drafting and editing. All authors approved the manuscript. 7. Funding This work is supported by grants from the Heart and Stroke Foundation of Canada (HSFC) (#G-23-0035047), and the Canadian Institutes of Health Research (CIHR) (#169062; #186148) (all to Ayman ElAli). Ayman ElAli holds a Tier 2 Canada Research Chair in molecular and cellular neurovascular interactions. Esther Trudel is the recipient of the scholarship of excellence Didier Mouginot of the Fondation du CHU de Québec – Desjardins . 8. Data and materials availability All other data are available from the corresponding author upon reasonable request. 9.1. Ethics approval: The experimental protocol was conducted in accordance with the ethical policies and procedures approved by the committee of animal protection of Université Laval (Comité de Protection des Animaux de l'Université Laval (CPAUL)-3; Approval # 22-1136). Handling of animals adhered to the principles outlined in the Canadian Council on Animal Care (CCAC) guidelines. Animal studies were reported according to ARRIVE 2.0 guidelines. 9.2. Consent to participate: Not applicable. 9.3. Competing interests: The authors declare no competing interests. 9.4. Clinical Trial Number: Not applicable. 9.5. Consent for publication: Not applicable. References Chojdak-Łukasiewicz J, Dziadkowiak E, Zimny A, Paradowski B. Cerebral small vessel disease: A review. Adv Clin Exp Med. 2021;30(3):349–56. 10.17219/acem/131216 . de Leeuw FE, de Groot JC, Achten E, et al. Prevalence of cerebral white matter lesions in elderly people: a population based magnetic resonance imaging study. The Rotterdam Scan Study. J Neurol Neurosurg Psychiatry. 2001;70(1):9–14. 10.1136/jnnp.70.1.9 . Petty GW, Brown RD, Whisnant JP, Sicks JD, O’Fallon WM, Wiebers DO. Ischemic stroke subtypes: a population-based study of functional outcome, survival, and recurrence. Stroke. 2000;31(5):1062–8. 10.1161/01.str.31.5.1062 . Rensma SP, van Sloten TT, Launer LJ, Stehouwer CDA. Cerebral small vessel disease and risk of incident stroke, dementia and depression, and all-cause mortality: A systematic review and meta-analysis. Neurosci Biobehav Rev. 2018;90:164–73. 10.1016/j.neubiorev.2018.04.003 . Wardlaw JM, Smith EE, Biessels GJ, et al. Neuroimaging standards for research into small vessel disease and its contribution to ageing and neurodegeneration. Lancet Neurol. 2013;12(8):822–38. 10.1016/S1474-4422(13)70124-8 . Pantoni L. Cerebral small vessel disease: from pathogenesis and clinical characteristics to therapeutic challenges. Lancet Neurol. 2010;9(7):689–701. 10.1016/S1474-4422(10)70104-6 . Wardlaw JM, Smith C, Dichgans M. Small vessel disease: mechanisms and clinical implications. Lancet Neurol. 2019;18(7):684–96. 10.1016/S1474-4422(19)30079-1 . Gorelick PB, Scuteri A, Black SE, et al. Vascular contributions to cognitive impairment and dementia: a statement for healthcare professionals from the american heart association/american stroke association. Stroke. 2011;42(9):2672–713. 10.1161/STR.0b013e3182299496 . Khan U, Porteous L, Hassan A, Markus HS. Risk factor profile of cerebral small vessel disease and its subtypes. J Neurol Neurosurg Psychiatry. 2007;78(7):702–6. 10.1136/jnnp.2006.103549 . Li Q, Yang Y, Reis C, et al. Cerebral Small Vessel Disease. Cell Transpl. 2018;27(12):1711–22. 10.1177/0963689718795148 . Silasi G, She J, Boyd JD, Xue S, Murphy TH. A mouse model of small-vessel disease that produces brain-wide-identified microocclusions and regionally selective neuronal injury. J Cereb Blood Flow Metab. 2015;35(5):734–8. 10.1038/jcbfm.2015.8 . Balbi M, Vanni MP, Vega MJ, et al. Longitudinal monitoring of mesoscopic cortical activity in a mouse model of microinfarcts reveals dissociations with behavioral and motor function. J Cereb Blood Flow Metab. 2019;39(8):1486–500. 10.1177/0271678X18763428 . Hermann DM, ElAli A. The Abluminal Endothelial Membrane in Neurovascular Remodeling in Health and Disease. Sci Signal. 2012;5(236). 10.1126/scisignal.2002886 . Menet R, Lecordier S, ElAli A. Wnt Pathway: An Emerging Player in Vascular and Traumatic Mediated Brain Injuries. Front Physiol. 2020;11:565667. 10.3389/fphys.2020.565667 . Marchetti B, Pluchino S. Wnt your brain be inflamed? Yes, it Wnt! Trends Mol Med. 2013;19(3):144–56. 10.1016/j.molmed.2012.12.001 . Mastroiacovo F, Busceti CL, Biagioni F, et al. Induction of the Wnt antagonist, Dickkopf-1, contributes to the development of neuronal death in models of brain focal ischemia. J Cereb Blood Flow Metab. 2009;29(2):264–76. 10.1038/jcbfm.2008.111 . Zhang QG, Wang R, Khan M, Mahesh V, Brann DW. Role of Dickkopf-1, an antagonist of the Wnt/beta-catenin signaling pathway, in estrogen-induced neuroprotection and attenuation of tau phosphorylation. J Neurosci. 2008;28(34):8430–41. 10.1523/JNEUROSCI.2752-08.2008 . Menet R, Bourassa P, Calon F, ElAli A. Dickkopf-related protein-1 inhibition attenuates amyloid-beta pathology associated to Alzheimer’s disease. Neurochem Int. 2020;141:104881. 10.1016/j.neuint.2020.104881 . Himburg HA, Doan PL, Quarmyne M, et al. Dickkopf-1 promotes hematopoietic regeneration via direct and niche-mediated mechanisms. Nat Med. 2017;23(1):91–9. 10.1038/nm.4251 . Seifert-Held T, Pekar T, Gattringer T, et al. Circulating Dickkopf-1 in acute ischemic stroke and clinically stable cerebrovascular disease. Atherosclerosis. 2011;218(1):233–7. 10.1016/j.atherosclerosis.2011.05.015 . He XW, Wang E, Bao YY, et al. High serum levels of sclerostin and Dickkopf-1 are associated with acute ischaemic stroke. Atherosclerosis. 2016;253:22–8. 10.1016/j.atherosclerosis.2016.08.003 . Zhu Z, Guo D, Zhong C, et al. Serum Dkk-1 (Dickkopf-1) Is a Potential Biomarker in the Prediction of Clinical Outcomes Among Patients With Acute Ischemic Stroke. Arterioscler Thromb Vasc Biol. 2019;39(2):285–93. 10.1161/ATVBAHA.118.311960 . Zhang K, Zhu Z, Shi M, et al. Serum Dickkopf-1 levels and poststroke depression in ischemic stroke patients. J Affect Disord. 2022;310:337–42. 10.1016/j.jad.2022.05.040 . Abraham HMA, Wolfson L, Moscufo N, Guttmann CRG, Kaplan RF, White WB. Cardiovascular risk factors and small vessel disease of the brain: Blood pressure, white matter lesions, and functional decline in older persons. J Cereb Blood Flow Metab. 2016;36(1):132–42. 10.1038/jcbfm.2015.121 . Cappuccio I, Calderone A, Busceti CL, et al. Induction of Dickkopf-1, a Negative Modulator of the Wnt Pathway, Is Required for the Development of Ischemic Neuronal Death. J Neurosci. 2005;25(10):2647–57. 10.1523/JNEUROSCI.5230-04.2005 . Xu H, Ding Z, Chen J, et al. Correlation between serum Dickkopf-1 (DKK1) levels and coronary artery stenosis. Nutr Metabolism Cardiovasc Dis. 2023;33(1):168–76. 10.1016/j.numecd.2022.10.007 . Lattanzio S, Santilli F, Liani R, et al. Circulating Dickkopf-1 in Diabetes Mellitus: Association With Platelet Activation and Effects of Improved Metabolic Control and Low‐Dose Aspirin. J Am Heart Assoc. 2014;3(4):e001000. 10.1161/JAHA.114.001000 . Lecordier S, Manrique-Castano D, El Moghrabi Y, ElAli A. Neurovascular Alterations in Vascular Dementia: Emphasis on Risk Factors. Front Aging Neurosci. 2021;13:727590. 10.3389/fnagi.2021.727590 . Volckaert T, Dill E, Campbell A, et al. Parabronchial smooth muscle constitutes an airway epithelial stem cell niche in the mouse lung after injury. J Clin Invest. 2011;121(11):4409–19. 10.1172/JCI58097 . Cvetkovic B, Yang B, Williamson RA, Sigmund CD. Appropriate tissue- and cell-specific expression of a single copy human angiotensinogen transgene specifically targeted upstream of the HPRT locus by homologous recombination. J Biol Chem. 2000;275(2):1073–8. 10.1074/jbc.275.2.1073 . Heaney JD, Rettew AN, Bronson SK. Tissue-specific expression of a BAC transgene targeted to the Hprt locus in mouse embryonic stem cells. Genomics. 2004;83(6):1072–82. 10.1016/j.ygeno.2003.12.015 . Seibenhener ML, Wooten MC. Use of the Open Field Maze to measure locomotor and anxiety-like behavior in mice. J Vis Exp. 2015;96e52434. 10.3791/52434 . Crawley J, Goodwin FK. Preliminary report of a simple animal behavior model for the anxiolytic effects of benzodiazepines. Pharmacol Biochem Behav. 1980;13(2):167–70. 10.1016/0091-3057(80)90067-2 . Bourin M, Hascoët M. The mouse light/dark box test. Eur J Pharmacol. 2003;463(1):55–65. 10.1016/S0014-2999(03)01274-3 . Al-Onaizi MA, Thériault P, Lecordier S, Prefontaine P, Rivest S, ElAli A. Early monocyte modulation by the non-erythropoietic peptide ARA 290 decelerates AD-like pathology progression. Brain Behav Immun. 2022;99:363–82. 10.1016/j.bbi.2021.07.016 . Thériault P, ElAli A, Rivest S. High fat diet exacerbates Alzheimer’s disease-related pathology in APPswe/PS1 mice. Oncotarget. 2016;7(42):67808–27. 10.18632/oncotarget.12179 . Bernard M, Menet R, Lecordier S, ElAli A. Endothelial PDGF-D contributes to neurovascular protection after ischemic stroke by rescuing pericyte functions. Cell Mol Life Sci. 2024;81(1):225. 10.1007/s00018-024-05244-w . Jean LeBlanc N, Menet R, Picard K, Parent G, Tremblay MÈ, ElAli A. Canonical Wnt Pathway Maintains Blood-Brain Barrier Integrity upon Ischemic Stroke and Its Activation Ameliorates Tissue Plasminogen Activator Therapy. Mol Neurobiol. 2019;56(9):6521–38. 10.1007/s12035-019-1539-9 . Thériault P, Le Béhot A, ElAli A, Rivest S. Sub-acute systemic erythropoietin administration reduces ischemic brain injury in an age-dependent manner. Oncotarget. 2016;7(24):35552–61. 10.18632/oncotarget.9652 . Yang Q, Wei X, Deng B, et al. Cerebral small vessel disease alters neurovascular unit regulation of microcirculation integrity involved in vascular cognitive impairment. Neurobiol Dis. 2022;170:105750. 10.1016/j.nbd.2022.105750 . Lecordier S, Pons V, Rivest S, ElAli A. Multifocal Cerebral Microinfarcts Modulate Early Alzheimer’s Disease Pathology in a Sex-Dependent Manner. Front Immunol. 2022;12:813536. 10.3389/fimmu.2021.813536 . Lecordier S, Menet R, Allain AS, ElAli A. Non-classical monocytes promote neurovascular repair in cerebral small vessel disease associated with microinfarctions via CX3CR1. J Cereb Blood Flow Metab. 2023;43(11):1873–90. 10.1177/0271678X231183742 . Seib DRM, Corsini NS, Ellwanger K, et al. Loss of Dickkopf-1 restores neurogenesis in old age and counteracts cognitive decline. Cell Stem Cell. 2013;12(2):204–14. 10.1016/j.stem.2012.11.010 . Clancy U, Gilmartin D, Jochems ACC, Knox L, Doubal FN, Wardlaw JM. Neuropsychiatric symptoms associated with cerebral small vessel disease: a systematic review and meta-analysis. Lancet Psychiatry. 2021;8(3):225–36. 10.1016/S2215-0366(20)30431-4 . Wang J, Zhu G, Huang L, et al. Morphine administration induces change in anxiety-related behavior via Wnt/β-catenin signaling. Neurosci Lett. 2017;639:199–206. 10.1016/j.neulet.2017.01.005 . Matrisciano F, Busceti CL, Bucci D, et al. Induction of the Wnt antagonist Dickkopf-1 is involved in stress-induced hippocampal damage. PLoS ONE. 2011;6(1):e16447. 10.1371/journal.pone.0016447 . Jayaraj RL, Azimullah S, Beiram R, Jalal FY, Rosenberg GA. Neuroinflammation: friend and foe for ischemic stroke. J Neuroinflammation. 2019;16(1):142. 10.1186/s12974-019-1516-2 . Candelario-Jalil E, Dijkhuizen RM, Magnus T, Neuroinflammation. Stroke, Blood-Brain Barrier Dysfunction, and Imaging Modalities. Stroke. 2022;53(5):1473–86. 10.1161/STROKEAHA.122.036946 . Park MH, Sung EA, Sell M, Chae WJ. Dickkopf1: An Immunomodulator in Tissue Injury, Inflammation, and Repair. ImmunoHorizons. 2021;5(11):898–908. 10.4049/immunohorizons.2100015 . Noz MP, Telgte A, ter, Wiegertjes K, et al. Pro-inflammatory Monocyte Phenotype During Acute Progression of Cerebral Small Vessel Disease. Front Cardiovasc Med. 2021;8. 10.3389/fcvm.2021.639361 . ElAli A, Jean LeBlanc N. The Role of Monocytes in Ischemic Stroke Pathobiology: New Avenues to Explore. Front Aging Neurosci. 2016;8:29. 10.3389/fnagi.2016.00029 . Noz MP, Ter Telgte A, Wiegertjes K, et al. Pro-inflammatory Monocyte Phenotype During Acute Progression of Cerebral Small Vessel Disease. Front Cardiovasc Med. 2021;8:639361. 10.3389/fcvm.2021.639361 . Lotfi N, Zhang GX, Esmaeil N, Rostami A. Evaluation of the effect of GM-CSF blocking on the phenotype and function of human monocytes. Sci Rep. 2020;10(1):1567. 10.1038/s41598-020-58131-2 . Wardlaw JM, Allerhand M, Doubal FN, et al. Vascular risk factors, large-artery atheroma, and brain white matter hyperintensities. Neurology. 2014;82(15):1331–8. 10.1212/WNL.0000000000000312 . Cannistraro RJ, Badi M, Eidelman BH, Dickson DW, Middlebrooks EH, Meschia JF. CNS small vessel disease. Neurology. 2019;92(24):1146–56. 10.1212/WNL.0000000000007654 . Ueland T, Otterdal K, Lekva T, et al. Dickkopf-1 enhances inflammatory interaction between platelets and endothelial cells and shows increased expression in atherosclerosis. Arterioscler Thromb Vasc Biol. 2009;29(8):1228–34. 10.1161/ATVBAHA.109.189761 . Cai L, Geng X, Hussain M, et al. Weight loss: indication of brain damage and effect of combined normobaric oxygen and ethanol therapy after stroke. Neurol Res. 2015;37(5):441–6. 10.1179/1743132815Y.0000000033 . Kim Y, Kim CK, Jung S, Ko SB, Lee SH, Yoon BW. Prognostic importance of weight change on short-term functional outcome in acute ischemic stroke. Int J Stroke. 2015;10(Suppl A):62–8. 10.1111/ijs.12554 . Wong SM, Jansen JFA, Zhang CE, et al. Blood-brain barrier impairment and hypoperfusion are linked in cerebral small vessel disease. Neurology. 2019;92(15):e1669–77. 10.1212/WNL.0000000000007263 . Schreiber S, Bueche CZ, Garz C, Braun H. Blood brain barrier breakdown as the starting point of cerebral small vessel disease? - New insights from a rat model. Experimental Translational Stroke Med. 2013;5(1):4. 10.1186/2040-7378-5-4 . KUNZ A, IADECOLA C. Cerebral vascular dysregulation in the ischemic brain. Handb Clin Neurol. 2009;92:283–305. 10.1016/S0072-9752(08)01914-3 . Johnson AC. Hippocampal Vascular Supply and Its Role in Vascular Cognitive Impairment. Stroke. 2023;54(3):673–85. 10.1161/STROKEAHA.122.038263 . Arvidsson A, Collin T, Kirik D, Kokaia Z, Lindvall O. Neuronal replacement from endogenous precursors in the adult brain after stroke. Nat Med. 2002;8(9):963–70. 10.1038/nm747 . Nakatomi H, Kuriu T, Okabe S, et al. Regeneration of hippocampal pyramidal neurons after ischemic brain injury by recruitment of endogenous neural progenitors. Cell. 2002;110(4):429–41. 10.1016/s0092-8674(02)00862-0 . Macas J, Nern C, Plate KH, Momma S. Increased generation of neuronal progenitors after ischemic injury in the aged adult human forebrain. J Neurosci. 2006;26(50):13114–9. 10.1523/JNEUROSCI.4667-06.2006 . Ekonomou A, Ballard CG, Pathmanaban ON, et al. Increased neural progenitors in vascular dementia. Neurobiol Aging. 2011;32(12):2152–61. 10.1016/j.neurobiolaging.2010.01.007 . Ciani L, Salinas PC. WNTs in the vertebrate nervous system: from patterning to neuronal connectivity. Nat Rev Neurosci. 2005;6(5):351–62. 10.1038/nrn1665 . Hattori Y, Enmi Jichiro, Kitamura A, et al. A novel mouse model of subcortical infarcts with dementia. J Neurosci. 2015;35(9):3915–28. 10.1523/JNEUROSCI.3970-14.2015 . Wang T, Chen Y, Zou Y, et al. Locomotor Hyperactivity in the Early-Stage Alzheimer’s Disease-like Pathology of APP/PS1 Mice: Associated with Impaired Polarization of Astrocyte Aquaporin 4. Aging Dis. 2022;13(5):1504–22. 10.14336/AD.2022.0219 . Low A, Mak E, Rowe JB, Markus HS, O’Brien JT. Inflammation and cerebral small vessel disease: A systematic review. Ageing Res Rev. 2019;53:100916. 10.1016/j.arr.2019.100916 . Neumann H, Kotter MR, Franklin RJM. Debris clearance by microglia: an essential link between degeneration and regeneration. Brain. 2009;132(Pt 2):288–95. 10.1093/brain/awn109 . Guruswamy R, ElAli A. Complex Roles of Microglial Cells in Ischemic Stroke Pathobiology: New Insights and Future Directions. Int J Mol Sci. 2017;18(3):496. 10.3390/ijms18030496 . Werner Y, Mass E, Ashok Kumar P, et al. Cxcr4 distinguishes HSC-derived monocytes from microglia and reveals monocyte immune responses to experimental stroke. Nat Neurosci. 2020;23(3):351–62. 10.1038/s41593-020-0585-y . Yang Y, Yao Z, Huo L. Causal effect of circulating cytokines on MRI markers of cerebral small vessel disease: A mendelian randomization study. Cytokine. 2024;182:156713. 10.1016/j.cyto.2024.156713 . Bhattacharya P, Budnick I, Singh M, et al. Dual Role of GM-CSF as a Pro-Inflammatory and a Regulatory Cytokine: Implications for Immune Therapy. J Interferon Cytokine Res. 2015;35(8):585–99. 10.1089/jir.2014.0149 . Spera PA, Ellison JA, Feuerstein GZ, Barone FC. IL-10 reduces rat brain injury following focal stroke. Neurosci Lett. 1998;251(3):189–92. 10.1016/s0304-3940(98)00537-0 . Yilmaz G, Arumugam TV, Stokes KY, Granger DN. Role of T Lymphocytes and Interferon-γ in Ischemic Stroke. Circulation. 2006;113(17):2105–12. 10.1161/CIRCULATIONAHA.105.593046 . Tables Additional Declarations No competing interests reported. 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09:48:00","extension":"html","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":213656,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7622603/v1/be7f4caa42045800ce6d7478.html"},{"id":94651651,"identity":"5f130960-2f46-4345-a532-9beeac9b6d1f","added_by":"auto","created_at":"2025-10-29 09:47:59","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":866742,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEndogenous DKK1 circulating levels are increased after cSVD.\u003c/strong\u003e (\u003cstrong\u003ea\u003c/strong\u003e) Analysis of the temporal regulation of DKK1 expression in the serum of C57BL6/J mice at baseline prior to cSVD, 24 hours, 14 and 30 days after cSVD. (\u003cstrong\u003eb\u003c/strong\u003e) A schematic illustration of the experimental design. (\u003cstrong\u003ec\u003c/strong\u003e) Analysis of DKK1 serum levels 14 days after cSVD in WT/DOX mice and DKK1/ON mice. (\u003cstrong\u003ed\u003c/strong\u003e) Analysis of DKK1 serum levels 30 days after cSVD in WT/DOX➞OFF and DKK1/ON➞OFF mice. (\u003cstrong\u003ee\u003c/strong\u003e) Analysis of weight loss as a percentage of baseline after cSVD in WT mice and DKK1 transgenic mice. Data are box plot with min/max (\u003cstrong\u003eb\u003c/strong\u003e, n=4-11 animals/group,\u003cstrong\u003e c\u003c/strong\u003e, \u003cstrong\u003ed\u003c/strong\u003e, n=11 animals/group, \u003cstrong\u003ee\u003c/strong\u003e, n=7-8 animals/group). *P\u0026lt;0.05/**P\u0026lt;0.01 (\u003cstrong\u003ec\u003c/strong\u003e, Mann-Whitney test; \u003cstrong\u003ee\u003c/strong\u003e, Repeated-measures two-way ANOVA followed by Tukey’s multiple comparisons tests). D, Days.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-7622603/v1/6f9b2c07d05b12bde827c8e4.png"},{"id":94651655,"identity":"12a27dc4-34ad-4093-9b04-4d0fa27ae09c","added_by":"auto","created_at":"2025-10-29 09:47:59","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3543734,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDKK1 elevated levels impair vascular integrity and reactivity after cSVD.\u003c/strong\u003e (\u003cstrong\u003ea\u003c/strong\u003e) Representative images of IgG immunolabeling in the whole brain and ipsilateral hippocampus of WT/DOX mice and DKK1/ON mice 14 days after cSVD. Analysis of ipsilateral/contralateral ratio of IgG intensity in the (\u003cstrong\u003eb\u003c/strong\u003e) whole hemisphere and (\u003cstrong\u003ec\u003c/strong\u003e) hippocampus of WT/DOX mice and DKK1/ON mice 14 days after cSVD. (\u003cstrong\u003ed\u003c/strong\u003e) Representative LSCI images of WT/DOX mice and DKK1/ON mice at baseline, 5 minutes and 7 days after cSVD. LSCI analysis of absolute brain perfusion in the (\u003cstrong\u003ee\u003c/strong\u003e) ipsilateral and (\u003cstrong\u003ef\u003c/strong\u003e) contralateral hemispheres in WT/DOX mice and DKK1/ON mice at baseline, 5 minutes and 7 days after cSVD. (\u003cstrong\u003eg\u003c/strong\u003e) LSCI analysis of brain perfusion in the ipsilateral hemisphere as a percentage of the contralateral hemisphere in WT/DOX mice and DKK1/ON mice at baseline, 5 minutes and 7 days after cSVD. Analysis of (\u003cstrong\u003eh\u003c/strong\u003e) laser Doppler flowmeter (LDF), and (\u003cstrong\u003ei\u003c/strong\u003e) velocity in the ipsilateral hemisphere as a percentage of the contralateral hemisphere in WT/DOX mice and DKK1/ON mice at baseline, 5 minutes and 7 days after cSVD. Data are box plot with min/max (\u003cstrong\u003eb\u003c/strong\u003e, \u003cstrong\u003ec\u003c/strong\u003e) and mean with SD (\u003cstrong\u003ee-i\u003c/strong\u003e) (n=7-8 animals/group). *P\u0026lt;0.05/**P\u0026lt;0.01 (\u003cstrong\u003eb\u003c/strong\u003e, Mann-Whitney test; \u003cstrong\u003ee\u003c/strong\u003e, \u003cstrong\u003eg\u003c/strong\u003e, \u003cstrong\u003eh\u003c/strong\u003e, \u003cstrong\u003ei\u003c/strong\u003e, Two-way ANOVA followed by Tukey’s multiple comparisons tests). D, Days, min, minutes. Scale bar = 500 mm and 200 mm (\u003cstrong\u003ea\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-7622603/v1/c950fd012f16a5fe82281b66.png"},{"id":94672857,"identity":"2e8d3780-1c59-45ff-803f-ae38eb604c59","added_by":"auto","created_at":"2025-10-29 13:41:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4367275,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDKK1 promotes neuronal loss and hampers neurogenesis after cSVD.\u003c/strong\u003e (\u003cstrong\u003ea\u003c/strong\u003e) Representative fluorescence images of NeuN\u003csup\u003e+\u003c/sup\u003e cells in the hippocampus 14 and 30 days after cSVD. Analysis of the area occupied by NeuN\u003csup\u003e+\u003c/sup\u003e CA regions in percentage of total contralateral and ipsilateral hippocampus area of (\u003cstrong\u003eb\u003c/strong\u003e) WT/DOX mice and (\u003cstrong\u003ec\u003c/strong\u003e) DKK1/ON mice 14 days after cSVD, (\u003cstrong\u003ed\u003c/strong\u003e) WT/DOX➞OFF mice and (\u003cstrong\u003ee\u003c/strong\u003e) DKK1/ON➞OFF mice 30 days after cSVD. (\u003cstrong\u003ef\u003c/strong\u003e) Representative fluorescence images of DCX\u003csup\u003e+\u003c/sup\u003e cells in the dentate gyrus 14 and 30 days after cSVD. Analysis of DCX intensity in the (\u003cstrong\u003eg\u003c/strong\u003e) contralateral and (\u003cstrong\u003eh\u003c/strong\u003e) ipsilateral dentate gyrus (DG) of WT/DOX and DKK1/ON mice 14 days after cSVD and of WT/DOX➞OFF and DKK1/ON➞OFF mice 30 days after cSVD. Analysis of DCX\u003csup\u003e+\u003c/sup\u003e cell projections length in the (\u003cstrong\u003ei\u003c/strong\u003e) contralateral and (\u003cstrong\u003ej\u003c/strong\u003e) ipsilateral DG of WT/DOX and DKK1/ON mice 14 days after cSVD and of WT/DOX➞OFF and DKK1/ON➞OFF mice 30 days after cSVD. Data are box plot with min/max (For 14 days timepoint n=7-8 animals/group, for 30 days timepoint n=7-10 animals/group). *P\u0026lt;0.05/**P\u0026lt;0.01 /***P\u0026lt;0.001/****P\u0026lt;0.0001 (\u003cstrong\u003eb\u003c/strong\u003e, \u003cstrong\u003ec\u003c/strong\u003e, \u003cstrong\u003eg\u003c/strong\u003e, \u003cstrong\u003eh\u003c/strong\u003e, \u003cstrong\u003ei\u003c/strong\u003e, \u003cstrong\u003ej\u003c/strong\u003e, unpaired t-test; \u003cstrong\u003ed\u003c/strong\u003e, \u003cstrong\u003ee\u003c/strong\u003e, Mann-Whitney test). D, days; C, contralateral; I, ipsilateral. Scale bar = 200 mm (\u003cstrong\u003ea\u003c/strong\u003e) and 50 mm (\u003cstrong\u003ef\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-7622603/v1/d9225f4f9746aca032a9ddeb.png"},{"id":94672219,"identity":"9c96f7dc-714f-4601-a238-d43b3f9e37b6","added_by":"auto","created_at":"2025-10-29 13:39:56","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1470604,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDKK1 overexpression induces hyperactivity independently of microinfarctions.\u003c/strong\u003e (\u003cstrong\u003ea\u003c/strong\u003e) A schematic illustration of the open field apparatus. Analysis of the (\u003cstrong\u003eb\u003c/strong\u003e) time spent in the center zone, (\u003cstrong\u003ec\u003c/strong\u003e) time spent in the corner zones, (\u003cstrong\u003ed\u003c/strong\u003e) total distance travelled and (\u003cstrong\u003ee\u003c/strong\u003e) freezing time of WT mice and DKK1 transgenic mice at baseline, 14 days after cSVD during DKK1 induction, and 30 days after cSVD upon normalization of its expression levels. (\u003cstrong\u003ef\u003c/strong\u003e) A schematic illustration of the light and dark box test apparatus. Analysis of the (\u003cstrong\u003eg\u003c/strong\u003e) latency of first entry in the light box, (\u003cstrong\u003eh\u003c/strong\u003e) number of entries in the light box, and (\u003cstrong\u003ei\u003c/strong\u003e) exploration time in the light box of WT mice and DKK1 transgenic mice at baseline, 14 days after cSVD during DKK1 induction, and at 30 days after cSVD upon normalization of its expression levels. (\u003cstrong\u003ej\u003c/strong\u003e) A schematic illustration of the NOR apparatus. Analysis of (\u003cstrong\u003ek\u003c/strong\u003e) total exploration time of both objects and (\u003cstrong\u003el\u003c/strong\u003e) the discrimination index of WT mice and DKK1 transgenic mice at baseline,14 days after cSVD during DKK1 induction, and 30 days after cSVD upon normalization of its expression levels. Data are mean with SD (\u003cstrong\u003eb, c\u003c/strong\u003e, \u003cstrong\u003ed\u003c/strong\u003e, \u003cstrong\u003ee\u003c/strong\u003e, \u003cstrong\u003eg\u003c/strong\u003e, \u003cstrong\u003eh\u003c/strong\u003e, \u003cstrong\u003ei\u003c/strong\u003e, \u003cstrong\u003ek\u003c/strong\u003e, n=7-10 animals/group, \u003cstrong\u003el\u003c/strong\u003e, n=6-9 animals/group). *P\u0026lt;0.05/**P\u0026lt;0.01 (\u003cstrong\u003ed\u003c/strong\u003e, \u003cstrong\u003ee\u003c/strong\u003e, Two-way ANOVA followed by Tukey’s multiple comparisons tests). D, days.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-7622603/v1/a57ca2bebdfab69ae0bc2b60.png"},{"id":94672227,"identity":"8b8b93f4-adbf-4820-80ca-57309f6a877d","added_by":"auto","created_at":"2025-10-29 13:39:57","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":6132528,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCellular neuroinflammation upon cSVD is minimally affected by DKK1 elevated levels.\u003c/strong\u003e (\u003cstrong\u003ea\u003c/strong\u003e) Representative fluorescence images of IBA-1\u003csup\u003e+\u003c/sup\u003e cells in the ipsilateral hemisphere and ipsilateral hippocampus 14 and 30 days after cSVD. Analysis of IBA-1 intensity in the (\u003cstrong\u003eb\u003c/strong\u003e) whole ipsilateral hemisphere and (\u003cstrong\u003ec\u003c/strong\u003e) ipsilateral hippocampus of WT/DOX and DKK1/ON mice 14 days after cSVD, and of WT/DOX➞OFF and DKK1/ON➞OFF mice 30 days after cSVD.\u0026nbsp; Analysis of reactive IBA-1\u003csup\u003e+\u003c/sup\u003e coverage in the (\u003cstrong\u003ed\u003c/strong\u003e) whole ipsilateral hemisphere and (\u003cstrong\u003ee\u003c/strong\u003e) ipsilateral hippocampus of WT/DOX and DKK1/ON mice 14 days after cSVD, and of WT/DOX➞OFF and DKK1/ON➞OFF mice 30 days after cSVD. (\u003cstrong\u003ef\u003c/strong\u003e) Representative fluorescence images of CD45\u003csup\u003e+\u003c/sup\u003e cells in the ipsilateral hemisphere 14 and 30 days after cSVD. Analysis of (\u003cstrong\u003eg\u003c/strong\u003e) ipsilateral/contralateral ratio of CD45 intensity in the whole hemisphere and (\u003cstrong\u003eh\u003c/strong\u003e) the mean gray value of CD45 signal in the ipsilateral hemisphere of WT/DOX and DKK1/ON mice 14 days after cSVD. Analysis of (\u003cstrong\u003ei\u003c/strong\u003e) the ipsilateral/contralateral ratio of CD45 intensity in the whole hemisphere and (\u003cstrong\u003ej\u003c/strong\u003e) the mean gray value of CD45 signal in the ipsilateral hemisphere of WT/DOX➞OFF and DKK1/ON➞OFF mice 30 days after cSVD. Data are box plot with min/max (n=7-10 animals/group). ***P\u0026lt;0.001 (\u003cstrong\u003ej\u003c/strong\u003e, Mann-Whitney test). D, days. Scale bar = 500 mm (\u003cstrong\u003ea, f\u003c/strong\u003e) and 200 mm (\u003cstrong\u003ea\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-7622603/v1/6fd0567e81d3cc0c0b145fef.png"},{"id":94651658,"identity":"f6c0950a-c1cd-4c48-9caf-ffbeca6850bd","added_by":"auto","created_at":"2025-10-29 09:47:59","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1946416,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDKK1 promotes pro-inflammatory monocyte polarization after cSVD.\u003c/strong\u003e (\u003cstrong\u003ea\u003c/strong\u003e) The gating strategy used to analyze the distribution of the different subsets of monocytes (CD11b\u003csup\u003e+\u003c/sup\u003eLy6C\u003csup\u003e+\u003c/sup\u003e) in blood circulation; patrolling (Ly6C\u003csup\u003elow\u003c/sup\u003e), intermediate (Ly6C\u003csup\u003einter\u003c/sup\u003e) and pro-inflammatory (Ly6C\u003csup\u003ehigh\u003c/sup\u003e) monocytes. Flow cytometry analysis of the frequency of (\u003cstrong\u003eb\u003c/strong\u003e) total leukocytes (CD45\u003csup\u003e+\u003c/sup\u003e), (\u003cstrong\u003ec\u003c/strong\u003e) total, (\u003cstrong\u003ed\u003c/strong\u003e) Ly6C\u003csup\u003elow\u003c/sup\u003e, (\u003cstrong\u003ee\u003c/strong\u003e) Ly6C\u003csup\u003einter\u003c/sup\u003e and (\u003cstrong\u003ef\u003c/strong\u003e) Ly6C\u003csup\u003ehigh\u003c/sup\u003e monocytes at baseline prior to cSVD in WT/DOX mice and DKK1/ON mice. Flow cytometry analysis of the frequency of (\u003cstrong\u003eg\u003c/strong\u003e) total leucocytes (CD45\u003csup\u003e+\u003c/sup\u003e), (\u003cstrong\u003eh\u003c/strong\u003e) total, (\u003cstrong\u003ei\u003c/strong\u003e) Ly6C\u003csup\u003elow\u003c/sup\u003e, (\u003cstrong\u003ej\u003c/strong\u003e) Ly6C\u003csup\u003einter\u003c/sup\u003e and (\u003cstrong\u003ek\u003c/strong\u003e) Ly6C\u003csup\u003ehigh\u003c/sup\u003e monocytes in WT/DOX mice and DKK1/ON mice 14 days after cSVD. Flow cytometry analysis of the frequency of (\u003cstrong\u003el\u003c/strong\u003e) total leucocytes (CD45\u003csup\u003e+\u003c/sup\u003e), (\u003cstrong\u003em\u003c/strong\u003e) total, (\u003cstrong\u003en\u003c/strong\u003e) Ly6C\u003csup\u003elow\u003c/sup\u003e, (\u003cstrong\u003eo\u003c/strong\u003e) Ly6C\u003csup\u003einter\u003c/sup\u003e and (\u003cstrong\u003ep\u003c/strong\u003e) Ly6C\u003csup\u003ehigh\u003c/sup\u003e monocytes in WT/DOX➞OFF and DKK1/ON➞OFF mice 30 days after cSVD. (\u003cstrong\u003eq\u003c/strong\u003e) A graph representing the distribution of monocyte subsets at baseline, 14 days and 30 days after cSVD of WT mice and DKK1 transgenic mice. Data are box plot with min/max (n=7-10 animals/group). ***P\u0026lt;0.001/****P\u0026lt;0.0001 (\u003cstrong\u003en\u003c/strong\u003e, \u003cstrong\u003eo\u003c/strong\u003e, \u003cstrong\u003ep\u003c/strong\u003e, Unpaired t-test). D, days.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-7622603/v1/c331df3cbc5758ba8f5f6ab5.png"},{"id":100070092,"identity":"049f9698-3553-43ff-a260-6ebdcdf9d90d","added_by":"auto","created_at":"2026-01-12 16:16:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":19167603,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7622603/v1/84fd1b82-e0e6-4bed-b96c-961f0f979427.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Dickkopf-1 accentuates neuronal damage upon multifocal cerebral microinfarctions by impairing vascular and immune responses","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eCerebral small vessel disease (cSVD) encompasses a diverse group of microangiopathies and represents the most common chronic and progressive cerebrovascular disease [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. cSVD affects nearly 80% of people over the age of 60, accounts for about 25% of ischemic stroke, often occurs with Alzheimer\u0026rsquo;s disease (AD), and is now recognized as a major contributor to vascular dementia (VaD) [\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The neuropathological features of cSVD are heterogenous and include subcortical microinfarcts, lacunar infarcts, microbleeds, enlarged perivascular spaces, white matter hyperintensities (WMH), and brain atrophy [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Depending upon the affected brain structure, distinct as well as common clinical manifestations occur, including stroke-related symptoms, cognitive decline, dementia, and psychiatric disorders [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The prevalence of cSVD is expected to continue rising over the next decade due to the ageing population and the high incidence of various vascular risk factors, including metabolic diseases, thereby imposing a significant socioeconomic burden [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. A better understanding of the mechanisms underlying the pathobiology of cSVD could improve patient management upon hospitalization and support the development of new effective preventative and therapeutic interventions.\u003c/p\u003e\u003cp\u003ecSVD associated with occlusion of small penetrating arterioles, leading to multifocal microinfarcts, represents a very common type [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The pathobiological processes essentially converge towards deregulation of neurovascular functions, inflammation, and secondary neurodegeneration [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Dickkopf-1 (DKK1) is a secreted glycoprotein that act as a potent inhibitor of the canonical Wnt pathway, which plays a major role in regulating neurovascular and immune functions [\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. While DKK1 expression is absent in the healthy brain, it becomes detectable under various vascular-related pathological conditions, including ischemia [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. DKK1 increased expression narrowly correlates with deregulation of canonical Wnt pathway activity [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. DKK1 is abundantly expressed in the blood circulation, presumably deriving from the bone marrow, where it plays an important role in regulating hematopoiesis [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Notably, DKK1 levels are elevated in the blood circulation of patients with acute and stable cerebrovascular diseases associated with large- as well as small-artery occlusion (SAO), correlating with poor prognosis [\u003cspan additionalcitationids=\"CR21 CR22\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Furthermore, DKK1 circulating levels are reported to be elevated in most of the leading risk factors of cSVD, mainly hypertension, hyperlipidemia, cardiovascular diseases and metabolic disorders [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan additionalcitationids=\"CR25 CR26 CR27\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. These observations suggest that DKK1 is potentially involved in modulating the pathobiology of cSVD associated with microinfarcts.\u003c/p\u003e\u003cp\u003eHerein, we aimed to investigate the role of DKK1 in affecting the pathobiology of cSVD associated with microinfarcts. This was achieved using a novel transgenic mouse model that enables a conditional widespread induction of DKK1 in a tissue-specific manner (i.e. inducible DKK1 mice). cSVD associated with microinfarcts was induced via sporadic micro-occlusion (MO) of cerebral penetrating arterioles using intravascularly injected microbeads (i.e. micro-emboli). Herein, we show that endogenous DKK1 levels immediately increase in the blood circulation following cSVD, remain elevated for several weeks, and return to basal levels at one month, outlining a prolonged, but transient upregulation. Our results reveal that DKK1 conditional widespread induction in respect to its endogenous temporal regulation after cSVD worsens vascular permeability as well as alters vascular reactivity translated by an impaired acute brain perfusion after micro-occlusions. These changes are associated with aggravated neuronal loss and cognitive deficits. Moreover, we demonstrate that DKK1 elevated levels in blood circulation deregulates the immune responses in the brain and periphery. The effects of DKK1 widespread prolonged induction are sufficient to mediate persistent long-term damage. Our study indicates that DKK1 elevated levels play a determinant role in the pathobiology of cSVD associated with microinfarcts via deregulation of neurovascular and immune functions, highlighting its potential to develop novel prognostic tools and therapeutic interventions.\u003c/p\u003e"},{"header":"2. Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Animal experiments\u003c/h2\u003e\u003cp\u003eThree to 5 months old transgenic mice [Gt(ROSA)26Sor\u003csup\u003etm1.1(rtTA,EGFP)Nagy\u003c/sup\u003e Hprt\u003csup\u003etm1(tetO\u0026minus;Dkk1)Spdl\u003c/sup\u003e Tg(TCF/Lef1-lacZ)34Efu/J; Stock N. 024746; The Jackson Laboratory] were used. The mice carry a knock-in allele containing \u003cem\u003eDkk1\u003c/em\u003e gene under the control of tetO (tetO-\u003cem\u003eDkk1\u003c/em\u003e), inserted into the endogenous \u003cem\u003eHprt\u003c/em\u003e locus on X chromosome [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. TetO-\u003cem\u003eDkk1\u003c/em\u003e transgene was inserted via homologous recombination, allowing the integration of a single copy into a specific location in the genome and subsequent tissue-specific expression of the transgene [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The mice carry a Gt(ROSA)26Sor\u003csup\u003etm1.1(rtTA,EGFP)Nagy\u003c/sup\u003e allele allowing a widespread expression of rtTA-EGFP as well as a Tg(TCF/Lef1-lacZ)34Efu reporter transgene. Widespread expression of \u003cem\u003eDkk1\u003c/em\u003e gene was induced using doxycycline for 14 days (in diet; 200 mg/kg, Bio-Serv, NJ, USA), which triggers a conformational switch in rtTA, allowing its binding to tetO. The strain was kept on C57BL6/J background and C57BL6/J wildtype (WT) mice are used as controls. The following nomenclature was adopted to identify the activation state of DKK1\u0026thinsp;=\u0026thinsp;DKK1/ON (transgenic mice with doxycycline), WT/DOX mice (WT mice with doxycycline), DKK1/ON\u0026rarr;OFF (DKK1/ON mice upon doxycycline withdrawal) and WT/DOX\u0026rarr;OFF (WT/DOX mice upon doxycycline withdrawal). Genotypes were confirmed by PCR as recommended by The Jackson Laboratory, using DNA isolated from ear punches and the following probes were used: \u003cem\u003eDkk1\u003c/em\u003e: Common: 5\u0026rsquo; \u0026ndash; GGA AAA TGG CTG TGG TCA GA \u0026ndash; 3\u0026rsquo;; Mutant forward: 5\u0026rsquo;- CCT ACC CTT CGC CTG AAG A \u0026ndash; 3\u0026rsquo;; Wildtype forward: 5\u0026rsquo;- AGA AAA GTC TCT ACT GCC TCC AC \u0026ndash; 3\u0026rsquo;, rTta: Wildtype reverse: 5\u0026rsquo; \u0026ndash; ATT CTG TGG GAA GTC TTG TCC \u0026ndash; 3\u0026rsquo;; Wildtype forward: 5\u0026rsquo; \u0026ndash; CTT CCC TCG TGA TCT GCA AC \u0026ndash; 3\u0026rsquo;; Mutant reverse: 5\u0026rsquo; \u0026ndash; GCT CTA ATG CGC TGT TAA TCA CT \u0026ndash; 3\u0026rsquo;; Mutant forward: 5\u0026rsquo; \u0026ndash; GGT ACC CGC TCG AAA TTC A \u0026ndash; 3\u0026rsquo;. All mice were housed under standard laboratory conditions and were provided with standard chow and water ad libitum. Animal experiments were conducted in accordance with the ethical policies and procedures approved by the committee of animal protection of Universit\u0026eacute; Laval (Comit\u0026eacute; de Protection des Animaux de l'Universit\u0026eacute; Laval (CPAUL)-3; Approval # 22-1136). Handling of animals adhered to the principles outlined in the Canadian Council on Animal Care (CCAC) guidelines. Animal studies were reported according to ARRIVE 2.0 guidelines.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 cSVD associated with microinfarcts model\u003c/h2\u003e\u003cp\u003eAdult male mice between 25\u0026ndash;30 g were subjected to micro-occlusions (MO) to generate cSVD associated with sporadic multifocal microinfarcts, as previously described [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Briefly, mice were anesthetized under 1.5% isoflurane in 1.5 l/minute (95% O2) and body temperature was maintained between 36 and 37\u0026deg;C using a feedback-controlled heating system (Harvard Apparatus, QC, Canada). A midline neck incision was performed to expose the left common carotid artery (CCA), the external carotid artery (ECA) and the pterygopalatine artery (PPA). The ECA and PPA were temporarily blocked using a microvascular clip under a surgical microscope (Leica Microsystems, ON, Canada) to ensure that all microspheres are directed towards the brain. Next, 2500 sterilized FITC-tagged microspheres of 20 \u0026micro;m (Polysciences Inc., PA, USA) suspended in 100 \u0026micro;L of PBS were slowly injected into the CCA using a 33G hypodermic needle (TSK Laboratory International, BC, Canada). Microspheres of 20 \u0026micro;m are used to occlude penetrating cerebral arterioles and capillaries [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. After injection, the ECA and PPA were unblocked, the needle was gently removed from the CCA, and the bleeding was stopped by applying pressure using bioabsorbable Gelfoam (Pfizer, NY, USA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Neurobehavioral analysis\u003c/h2\u003e\u003cp\u003eAnimals underwent neurobehavioral tests prior to surgery (baseline) and at 14 and 30 days after cSVD onset. \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eThe open field (OF) test\u003c/span\u003e was used to assess anxiety-like behavior and general locomotor ability as previously described [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Mice were individually placed in an open arena (45 cm x 45 cm) and were allowed to move freely for 10 minutes. Animals\u0026rsquo; movements were recorded, tracked, and analyzed using a video-tracking system (ANY-maze, Stoelting Co, IL, USA) in which the arena was divided into different zones namely, the center and corners. The parameters evaluated were the time spent in the center, the time spent in the corners, the total distance travelled and the total freezing time. An increase in the time spent in the center would indicate a disinhibitory behavior whereas a decrease shows an anxious state. A decrease in the distance could either mean a freezing state, a typical anxiety behavior, or motor impairment.\u003c/p\u003e\u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eThe light/dark box test\u003c/span\u003e was used to assess anxiety-like behavior in rodents. The test is based on rodent\u0026rsquo;s innate aversion to brightly illuminated areas and on the spontaneous exploratory behavior of rodents in response to mild stressors, such as light [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The apparatus is composed of a compartment exposed to light and a dark comportment, separated by a wall with an open door. Each animal is first placed in the dark compartment and allowed to freely explore both compartments for 5 minutes. Latency to the first entry into the light compartment, time spent in the light compartment and the number of entries into the light compartment were recorded and analyzed. A reduced latency to enter the lightened compartment is interpreted as anxiety-like behavior in mice [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eThe novel object recognition (NOR) test\u003c/span\u003e was used to assess recognition memory, as previously described [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Briefly, during the acquisition phase, mice were individually allowed to explore two identical objects in an open arena (45 cm \u0026times; 45 cm) for 10 minutes. Mice were returned to home cage for 1 hour and were placed back afterwards into the same arena, in the presence of one familiar object along with one novel object. Animals\u0026rsquo; movements were objectively monitored and quantified with ANY-maze. Object exploration was defined as touching the object or directing the nose toward it at \u0026lt;\u0026thinsp;2 cm. We evaluated the time spent on both objects to assess mice performances. The arena and objects were cleaned with 70% alcohol between each mouse and trial. Animals whose exploration time was lower than 10 seconds were not used for analysis. A discrimination index (DI) ratio was calculated as follows = (time novel object\u0026thinsp;\u0026minus;\u0026thinsp;time familiar object)/(time novel object\u0026thinsp;+\u0026thinsp;time familiar object). DI ranges from +\u0026thinsp;1 to \u0026minus;\u0026thinsp;1; a positive ratio indicates a preference for the novel object, a negative ratio indicates more time spent with the familiar object, and a ratio of zero reflects no preference [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Brain perfusion imaging and analysis\u003c/h2\u003e\u003cp\u003eBrain perfusion, which reflects the regional changes in cerebral blood flow (CBF), was investigated using Laser Speckle contrast imaging (LSCI) and laser Doppler flowmeter (LDF), as previously described [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Analysis was performed in the same animals to enable temporal assessment of brain perfusion at baseline, 5 minutes upon cSVD onset (immediate vascular responses) and on day 7 (delayed vascular responses). Briefly, mice were anesthetized using 1.5% isoflurane in 1.5 l/min (95% O2). Mice head was shaved, 100 \u0026micro;L of lidocaine/bupivacaine solution was applied on the incision site and the ears (50 \u0026micro;L/ear). Mice were placed on a stereotaxic frame (RWD Life Science Inc., CA, USA), the skull was exposed by removing the skin using fine-tip forceps, and brain perfusion was visualized using 2D laser Speckle blood flow imager (OMEGAZONE OZ-3, Omegawave Inc., Japan). The OZ-3 system is equipped with a visible and near infra-red (NIR) CCD camera, continuously displaying real images while comparing color differences between real and blood flow images. Brain perfusion in each hemisphere was calculated by averaging 20 consecutive raw Speckle images in each ROI using a built-in software. The ratio of blood perfusion in the ipsilateral over contralateral hemisphere was calculated. Brain perfusion was further analyzed using LDF (OMEGAFLOW FLO-N1; Omegawave Inc.). A non-contact fiber optic probe was positioned at 1 cm distance from the top of the skull covering the ipsilateral and contralateral hemisphere. The mean values of blood flow (i.e. brain perfusion), velocity, and mass were recorded for 20 seconds for each ROI and processed using a built-in software.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Brain tissue processing\u003c/h2\u003e\u003cp\u003eMice were euthanized 14 days and 1 month after cSVD onset. Briefly, mice were deeply anesthetized with a solution containing ketamine (90 mg/ml) and xylazine (10 mg/ml) then euthanized via transcardiac perfusion with PBS followed by 1% paraformaldehyde (PFA). Brains were extracted and post-fixed in 4% PFA for 24 hours followed by 4% PFA with 20% sucrose for 24 hours. Brains were cut into 25 \u0026micro;m coronal sections on microtome (Leica Biosystems, ON, Canada), and sections were collected in 12 well-plates containing an antifreeze solution (30% glycerol, 30% ethylene glycol in 0.9% sodium chloride (NaCl), phosphate buffer (PB)) and kept at -20\u0026deg;C for further use.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Immunofluorescence analysis\u003c/h2\u003e\u003cp\u003eFree-floating brain sections were first rinsed 3x in KPBS for 10 minutes and then incubated for 1 hour in a permeabilization/blocking solution containing 4% normal donkey serum (NDS) (Sigma-Aldrich, D9663), 1% bovine serum albumin (BSA) (Wisent Inc., 800-095-CG), and 1% Triton X-100 (Fisher scientific, BP151-100) in KPBS. Brain sections were then incubated overnight at room temperature with different primary antibodies diluted in the same solution. The following primary antibodies were used; chicken anti-NeuN (1:1000, MilliporeSigma, ABN91), rabbit anti-doublecortin (DCX) (1:1000, Abcam, ab207175), rabbit anti-ionized calcium binding adaptor molecule (IBA-1) (1:500, WAKO, 019-19741), rat anti-CD45 (1:500, BD Biosciences, 553076). The next day, brain sections were rinsed 3x in KPBS for 10 minutes and incubated for 2 hours at room temperature with the adequate secondary antibodies at a 1:700 dilution in KPBS; Cy3 donkey anti-chicken (Millipore Sigma, AP194C), Alexa Fluor 647 donkey anti-rabbit (Invitrogen, A31574), Alexa Fluor 647 donkey anti-rat (Jackson ImmunoResearch Laboratories, 712-605-153). Brain sections were then rinsed 3x with KPBS and incubated with 4\u0026prime;,6-diamidino-2-phenylindole (DAPI) (1:10 000) for 5 minutes. Brain sections were rinsed with KBPS, mounted onto Superfrost\u0026reg; micro-slides, and cover-slipped with Fluoromount-G\u0026reg; anti-fade medium (Electron Microscopy Science, 17984-25). Epifluorescence images were acquired using Axio Observer microscope equipped with optical sectioning (Apotome.2) and Axiocam 503 monochrome camera and processed using with ZEN Imaging Software (Carl Zeiss Canada, ON, Canada). NeuN\u0026thinsp;+\u0026thinsp;area in the hippocampus, DCX intensity and projection length, IBA-1 area and intensity and CD45 intensity were measured with FIJI software.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Immunohistochemical analysis\u003c/h2\u003e\u003cp\u003eImmunohistochemical analysis was used to assess extravasation of blood-borne immunoglobulin G (IgG), an endogenous marker of BBB breakdown, as previously described [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Brain sections were rinsed with KPBS (Sigma-Aldrich) and then incubated for 20 minutes in a permeabilization/blocking solution containing 4% normal goat serum (NGS; Sigma-Aldrich, GG9023), 1% BSA, and 0.4% Triton X-100 in KPBS. Brain sections were incubated overnight at 4\u0026deg;C with biotinylated goat anti-mouse IgG antibody (1/1000; BA-9200; Vector laboratories) in the same permeabilization/blocking solution. The next day, brain sections were rinsed 3x with KPBS and then incubated for 1 hour at room temperature with Avidin Biotin Peroxidase Complex (ABC) (Vectastain Elite Kit Standard). Brain sections were rinsed 3x with KPBS before incubation with 3,3\u0026rsquo;-diaminobenzidine tetrahydrochloride (DAB, Sigma-Aldrich) for 10 minutes, washed 3x with KPBS. Brain sections were mounted onto slides, dried, dehydrated via immersion in an increased concentration of ethanol (EthO) solution (50%, 70%, 75%, 95% and 100%), and finally immersed in Xylene solution 3x for 3 minutes and cover-slipped using DPX mounting solution (Electron Microscopy Sciences). Mounted sections were digitized and the intensity of DAB signal (optical density) in the ipsilateral and contralateral (i.e. background) cortex and hippocampus was analyzed using FIJI software. Ratio of ipsilateral/contralateral intensity was computed and values\u0026thinsp;\u0026gt;\u0026thinsp;1 indicates an increased IgG intensity (i.e. infiltration) in the ipsilateral brain structures.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8 Enzyme-linked immunosorbent assay (ELISA)\u003c/h2\u003e\u003cp\u003eMouse DKK1 Quantikine ELISA Kit (R\u0026amp;D Systems, MKK100) was used to quantify DKK1 concentration in the serum at 14 and 30 days after cSVD. Briefly, blood samples were collected from the submandibular vein and left at room temperature for 2 hours to allow clotting. The samples were then centrifuged at 2000 g for 20 minutes, after which the serum was separated, aliquoted, and stored at \u0026minus;\u0026thinsp;20\u0026deg;C until analysis. All samples were assayed in duplicates. Serum samples of DKK1/ON mice were diluted 1:50 and serum samples of WT/DOX, WT/DOX\u0026rarr;OFF and DKK1/ON\u0026rarr;OFF were diluted 1:10. The optical density was determined using a microplate reader (SpectraMax i3, Molecular Devices, CA, USA) at 450 nm with wavelength correction at 540 nm and analyzed using SOFTmax Pro 6.4.0.1 software (Molecular Devices). All experiments were performed according to the manufacturer's instructions (R\u0026amp;D Systems, MKK100).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9 Cytokine/chemokine array\u003c/h2\u003e\u003cp\u003eThe cytokine and chemokine concentrations in the serum of WT/DOX\u0026rarr;OFF and DKK1/ON\u0026rarr;OFF at 30 days after cSVD were analyzed at Eve Technologies (Calgary, Canada) using the Mouse Cytokine Proinflammatory Focused 10-Plex Discovery Assay Array (Eve Technologies, MDF10).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.10 Flow cytometry analysis\u003c/h2\u003e\u003cp\u003eBlood samples were collected from the submandibular vein into ethylene-diamine-tetra-acetic acid (EDTA) coated vials (Sarstedt, Montr\u0026eacute;al, QC, Canada). Analysis of the dynamics of monocytes was performed as previously described [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Briefly, 60 \u0026micro;L of total blood was incubated at room temperature for 5 minutes with 1 mL of ACK (ammonium, chloride, potassium) lysing buffer to get rid of red blood cells. Samples were then washed with 3 mL of PBS and centrifuge at 400 g for 8 minutes at 4\u0026deg;C. The supernatant was removed, and cells were resuspended with blockage solution (1 \u0026micro;L CD16/CD32 antibody (BD bioscience) diluted in 100 \u0026micro;L of PBS per tube) and incubated for 10 minutes on ice. Then, samples were incubated for 30 minutes in the dark on ice with 100 \u0026micro;L of the following antibody mix: cluster of differentiation (CD)-45 BV605 (1:400, Biolegend, 103139), CD11b Pe-Cy7 (1:100, EBioscience, 25\u0026ndash;0112), CD115 APC (1:75, EBioscience, 17-1152), Ly6C V450 (1:100, BD Bioscience, 560594), Ly6G Pe (1:200, BD bioscience, 551461) and live/dead AF 780 (1:400, BD bioscience, 565388). After incubation, the samples were washed with 3 mL PBS, centrifuged for 8 minutes at 400 g, resuspended in 250 \u0026micro;L of PBS, and complemented with 50 \u0026micro;L of 123count eBeads\u0026trade; Counting Beads (Invitrogen, 01-1234-42). Samples were processed using a LSR II flow cytometer, and data was acquired using BD FACS Diva software (Version 6.1.2, BD Bioscience), and analyzed using FlowJo software v10 (Tree Star; Ashland, OR, USA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.11 Statistical analysis\u003c/h2\u003e\u003cp\u003eData are presented as boxplot with min/max whiskers or mean \u0026plusmn; standard deviation (SD). Data distribution normality was assessed with the Shapiro Wilk test. If data followed normal distribution, unpaired two-tailed t-test was used for comparison between two groups, and one-way or two-way analysis of variance (ANOVA) followed by Tukey\u0026rsquo;s post-hoc test were used for multiple comparison. If data did not follow normal distribution, Mann-Whitney test was used for comparison between two groups and Kruskal-Wallis test followed by Dunn\u0026rsquo;s test was used for multiple comparison. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant. Statistical analyses were carried out using GraphPad Prism (version 10, GraphPad Software, San Diego, CA, USA).\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.1. cSVD is associated with a prolonged increase in DKK1 expression in blood circulation\u003c/h2\u003e\u003cp\u003ePrevious reports have indicated that circulating DKK1 levels are elevated in various vascular pathologies, including stroke [\u003cspan additionalcitationids=\"CR21 CR22\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], as well as vascular risk factors [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan additionalcitationids=\"CR25 CR26 CR27\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Herein, we aimed first to investigate the temporal regulation of DKK1 expression in blood circulation upon cSVD associated with microinfarcts. Our ELISA analysis showed that DKK1 levels in serum were rapidly increased in C57BL6/J mice upon cSVD at 24 hours [+\u0026thinsp;6.26%] and remained elevated until day 14 to subsequently return to basal levels at day 30 after onset [-17.86%] \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e. This suggests that cSVD onset is associated with a prolonged release of DKK1 in blood circulation that persists over time before normalization. To further characterize the role of DKK1, C57BL6/J mice (WT/DOX) and inducible DKK1 transgenic mice (DKK1/ON) on doxycycline (200 mg/kg; 14 days) were subjected to cSVD. In one cohort of mice, doxycycline was kept for 14 days after onset to maintain DKK1 induction, while in another cohort, doxycycline was withdrawn between day 14 and day 30 after onset, to normalize DKK1 expression (WT/DOX\u0026rarr;OFF, DKK1/ON\u0026rarr;OFF) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e. Using ELISA, we showed that DKK1 circulating levels at day 14 after cSVD were significantly induced in DKK1/ON mice (127 375.4 \u0026plusmn; 128 216.2 pg/mL) compared to WT/DOX mice (20 118.6 \u0026plusmn; 3315.2 pg/mL) [P\u0026thinsp;=\u0026thinsp;0.0014] \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec\u003cb\u003e)\u003c/b\u003e. Upon doxycycline withdrawal, DKK1 circulating levels at day 30 were similar in DKK1/ON\u0026rarr;OFF mice (14 162.2 \u0026plusmn; 4923.4 pg/mL) and WT/DOX\u0026rarr;OFF mice (16 135.1 \u0026plusmn; 5431.2 pg/mL) [P\u0026thinsp;=\u0026thinsp;0.3653] \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed\u003cb\u003e)\u003c/b\u003e. Moreover, DKK1 elevated levels significantly exacerbated weight loss in DKK1/ON mice compared to WT/DOX mice at day 3 [P\u0026thinsp;=\u0026thinsp;0.0452], day 4 [P\u0026thinsp;=\u0026thinsp;0.0111] and day 6 [P\u0026thinsp;=\u0026thinsp;0.0325] after cSVD \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee\u003cb\u003e)\u003c/b\u003e. Upon normalization of DKK1 levels, DKK1/ON\u0026rarr;OFF mice still had greater weight loss at day 30 after cSVD to WT/DOX\u0026rarr;OFF [P\u0026thinsp;=\u0026thinsp;0.0058] \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee\u003cb\u003e)\u003c/b\u003e. Our findings indicate that cSVD is associated with prolonged transient increase in DKK1 levels in blood circulation, correlating with an exacerbated alteration of the overall physical state of mice.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Excessive DKK1 circulating levels exacerbate structural and functional vascular integrity\u003c/h2\u003e\u003cp\u003eDeregulation of neurovascular functions, associated with alteration of the vascular permeability and cerebral microcirculation, play a central role in the pathobiology of cSVD [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. We first assessed the impact of DKK1 circulating elevated levels on vascular permeability via analysis blood-borne IgG extravasation into the brain 14 days after cSVD (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). IgG infiltration was higher in the ipsilateral hemisphere of DKK1/ON mice compared to WT/DOX mice [P\u0026thinsp;=\u0026thinsp;0.0541] \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e, which was less prominent in the hippocampus [P\u0026thinsp;=\u0026thinsp;0.1520] \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec\u003cb\u003e)\u003c/b\u003e, probably due to the sporadic micro-occlusions that generate spatially heterogenous microinfarcts in restricted brain regions. Next, we investigated the consequences of DKK1 induction on temporal brain perfusion upon cSVD using laser Speckle contrast imaging (LSCI) and non-contact laser Doppler flowmetry (LDF). LSCI was performed at baseline prior to cSVD, 5 minutes upon micro-occlusions (immediate impact of vascular reactivity) and 7 days (delayed vascular reactivity) after cSVD onset in WT/DOX and DKK1/ON mice \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed\u003cb\u003e)\u003c/b\u003e. LSCI analysis revealed that DKK1 induction potently reduced brain perfusion immediately (5 minutes) upon cSVD onset in the ipsilateral hemisphere [P\u0026thinsp;=\u0026thinsp;0.0066] \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee\u003cb\u003e)\u003c/b\u003e but not in the contralateral hemisphere [P\u0026thinsp;=\u0026thinsp;0.1466] \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef\u003cb\u003e)\u003c/b\u003e of DKK1/ON mice compared to WT/DOX mice. Brain perfusion was completely recovered in the ipsilateral [P\u0026thinsp;=\u0026thinsp;0.1872] and contralateral [P\u0026thinsp;=\u0026thinsp;0.6848] hemispheres of WT/DOX and DKK1/ON mice at day 7 after cSVD \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee, f\u003cb\u003e)\u003c/b\u003e. Furthermore, our analysis showed that DKK1 elevated levels are associated with an overall greater attenuation of brain perfusion immediately (5 minutes) upon cSVD onset in the ipsilateral hemisphere relatively to the contralateral hemisphere of DKK1/ON mice compared to WT/DOX mice [P\u0026thinsp;=\u0026thinsp;0.0263] \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg\u003cb\u003e)\u003c/b\u003e. This suggests that DKK1 elevated levels impair acute vascular reactivity in response to micro-occlusions, leading to an exacerbated reduction of cerebral microcirculation. These results were corroborated with LDF analysis that has outlined a significant reduction in blood flow [P\u0026thinsp;=\u0026thinsp;0.0267] \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh\u003cb\u003e)\u003c/b\u003e as well as velocity [P\u0026thinsp;=\u0026thinsp;0.0362] \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ei\u003cb\u003e)\u003c/b\u003e immediately (5 minutes) after cSVD in the ipsilateral hemisphere relatively to the contralateral hemisphere of DKK1/ON mice compared to WT/DOX mice. Our findings show that DKK1 elevated levels exacerbate vascular structural integrity increasing permeability and alter acute vascular reactivity, thereby impairing brain perfusion after cSVD.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.3. DKK1 induction aggravates neuronal loss and impairs neurogenesis in the hippocampus\u003c/h2\u003e\u003cp\u003eThe hippocampus, which plays a major role in various cognitive functions such as anxiety and memory, is highly vulnerable to ischemic injury [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. It has been previously shown that cSVD mediates neurodegenerative responses in the hippocampus [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Herein, we aimed to evaluate the impact of elevated circulating DKK1 levels on neuronal damage after cSVD particularly in the hippocampus. NeuN immunolabelling in the hippocampus at 14 and 30 days after cSVD was used to assess neuronal loss \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e. Our analysis at 14 days after cSVD indicated that WT/DOX mice displayed a significant reduction of NeuN\u003csup\u003e+\u003c/sup\u003e area in the CA of the ipsilateral hippocampus when compared to the area of matched intact CA of the contralateral hemisphere [P\u0026thinsp;=\u0026thinsp;0.0043] \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e. However, the reduction was more important in DKK1/ON mice [P\u0026thinsp;=\u0026thinsp;0.0002], indicating DKK1 elevated levels led to greater neuronal loss in the CA region \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec\u003cb\u003e)\u003c/b\u003e. Evaluation of neuronal loss at 30 days showed that WT/DOX\u0026rarr;OFF mice still exhibited an important reduction in NeuN\u003csup\u003e+\u003c/sup\u003e area in the CA of the ipsilateral hippocampus in comparison to the matched intact CA of the contralateral hemisphere [P\u0026thinsp;=\u0026thinsp;0.0006] \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed\u003cb\u003e)\u003c/b\u003e, which was less prominent in DKK1/ON\u0026rarr;OFF mice [P\u0026thinsp;=\u0026thinsp;0.0433] (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). This suggests that normalization of DKK1 levels upon doxycycline withdrawal prevents further neuronal loss after cSVD, and that the extent of neuronal loss is solely attributed to micro-occlusions. Next, we assessed the neurogenic response in the dentate gyrus (DG) by evaluating the turnover of neuronal progenitor cells (NPCs) using doublecortin (DCX) immunolabelling (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef). We first investigated basal neurogenesis in the contralateral DG, and observed that DCX mean intensity, indicative of the overall density of NPCs, was similar between WT/DOX and DKK1/ON mice at day 14 after cSVD (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg). However, DCX mean intensity was significantly reduced in the contralateral DG of DKK1/ON\u0026rarr;OFF mice compared to WT/DOX\u0026rarr;OFF mice [P\u0026thinsp;=\u0026thinsp;0.0003] at day 30 after cSVD (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg). On the other hand, DCX mean intensity was increased in WT/DOX mice compared to DKK1/ON mice in the ipsilateral DG at day 14 after cSVD [P\u0026thinsp;=\u0026thinsp;0.0128], which was maintained upon normalization of DKK1 expression in WT/DOX\u0026rarr;OFF mice compared to DKK1/ON\u0026rarr;OFF mice at day 30 days [P\u0026thinsp;=\u0026thinsp;0.0197] (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh). Next, we assessed the length of DCX\u003csup\u003e+\u003c/sup\u003e cell projections, indicative of overall dendritic integrity [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. In the contralateral DG, the mean length of DCX\u003csup\u003e+\u003c/sup\u003e cell projections was greater in DKK1/ON mice compared to WT/DOX mice at day 14 after cSVD [P\u0026thinsp;=\u0026thinsp;0.0401] but was significantly reduced in DKK1/ON\u0026rarr;OFF mice compared to WT/DOX\u0026rarr;OFF mice at day 30 [P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001] (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ei). In the ipsilateral DG, the mean length of DCX\u003csup\u003e+\u003c/sup\u003e cell projections remained unchanged at day 14 after cSVD between WT/DOX and DKK1/ON mice but was significantly reduced in DKK1/ON\u0026rarr;OFF mice compared to WT/DOX\u0026rarr;OFF at day 30 after cSVD [P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001] (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ej). Our results suggest that DKK1 excessive release at cSVD onset mediates a progressive impairment of neurogenesis despite delayed normalization of expression levels.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.4. Excessive DKK1 expression promotes hyperactive behavior independently upon cSVD\u003c/h2\u003e\u003cp\u003ecSVD contributes to cognitive decline and neuropsychiatric symptoms [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Furthermore, various reports have indicated that DKK1 is implicated in the pathological processes underlying stress-related disorders [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Hence, we investigated the impact of elevated DKK1 circulating levels after cSVD on anxiety-like behaviors and general ambulatory ability using the open field test (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). For time spent in the center, two-way analysis of variance (ANOVA) revealed no interaction between DKK1 induction state (WT mice and inducible DKK1 transgenic mice with or without DOX) and time associated with cSVD onset (baseline, 14 days, 30 days) [P\u0026thinsp;=\u0026thinsp;0.6233] (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). However, the main effect of time associated with cSVD onset was significant [P\u0026thinsp;=\u0026thinsp;0.0009] while the main effect of DKK1 induction state was not [P\u0026thinsp;=\u0026thinsp;0.2275], indicating the time spent in the center was affected essentially by cSVD, regardless of DKK1 levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). For time spent in the corners, two-way ANOVA revealed no interaction between DKK1 induction state and time associated with cSVD onset [P\u0026thinsp;=\u0026thinsp;0.5276] (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). Similarly, the main effect of time associated with cSVD onset was significant [P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001] while the main effect of DKK1 induction state was not [P\u0026thinsp;=\u0026thinsp;0.9525], suggesting that time spent in corners was affected mainly by cSVD, independently upon DKK1 levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). Although there was still no interaction for the total distance travelled [P\u0026thinsp;=\u0026thinsp;0.1895], the main effect of time associated with cSVD onset [P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001] as well DKK1 induction state [P\u0026thinsp;=\u0026thinsp;0.004] were both significant, indicating that the total distance travelled in the open field was affected by cSVD as well as DKK1 elevated levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). Tukey\u0026rsquo;s post-hoc test revealed that DKK1 induction increased overall mobility, translated by a more important distance travelled at baseline [P\u0026thinsp;=\u0026thinsp;0.0015] and 14 days after cSVD [P\u0026thinsp;=\u0026thinsp;0.0203] in inducible DKK1 transgenic mice compared to WT mice with DOX. Notably, there was no difference 30 days after cSVD between WT and inducible DKK1 mice without DOX [P\u0026thinsp;=\u0026thinsp;0.4393] (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed), suggesting that mobility was restored upon normalization of DKK1 levels. For the freezing time, two-way ANOVA showed no interaction between the two factors [P\u0026thinsp;=\u0026thinsp;0.3897], but the main effect of time associated with cSVD [P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001] and DKK1 induction state [P\u0026thinsp;=\u0026thinsp;0.0027] were significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). Tukey\u0026rsquo;s post-hoc test showed that inducible DKK1 transgenic mice with DOX exhibited reduced freezing time at day 14 [P\u0026thinsp;=\u0026thinsp;0.0194] and day 30 after cSVD [P\u0026thinsp;=\u0026thinsp;0.0215] (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). This indicates that while DKK1 induction did not affect the time spent in the different zones of the open field, it increased the total distance travelled and reduced the freezing time, outlining the emergence of hyperactive behavior. We further explored the anxiety-like behavior using the light and dark box test (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef). Two-way ANOVA of the latency of first entry in the light box showed no interaction between DKK1 induction state and time associated with cSVD onset [P\u0026thinsp;=\u0026thinsp;0.7658], in absence of independent main effects of DKK1 induction state [P\u0026thinsp;=\u0026thinsp;0.3479] or time associated with cSVD onset [P\u0026thinsp;=\u0026thinsp;0.3954] (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg). Similar results were obtained for the number of entries in the light box with no significant interaction between the two factors [P\u0026thinsp;=\u0026thinsp;0.2613], in absence of independent main effects of DKK1 induction state [P\u0026thinsp;=\u0026thinsp;0.4436] or time associated with cSVD onset [P\u0026thinsp;=\u0026thinsp;0.6055] (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh). There was no interaction between DKK1 induction state and time associated with cSVD onset on the total exploration time in the light box [P\u0026thinsp;=\u0026thinsp;0.9891], with no independent main effects of DKK1 induction state [P\u0026thinsp;=\u0026thinsp;0.1731] or time associated with cSVD onset [P\u0026thinsp;=\u0026thinsp;0.2614] (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ei). These results suggest neither time after cSVD nor DKK1 elevated levels significantly influenced anxiety-like behaviors in mice in the light and dark box test. Next, we used novel object recognition (NOR) test to assess recognition memory (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ej). Two-way ANOVA revealed no interaction between DKK1 induction state and time associated with cSVD onset for the total exploration time of both objects [P\u0026thinsp;=\u0026thinsp;0.7953], but the main effect of time associated with cSVD onset was significant [P\u0026thinsp;=\u0026thinsp;0.0249], indicating that the innate instinct to explore objects varied over time after cSVD, independently of the main effect of DKK1 induction state [P\u0026thinsp;=\u0026thinsp;0.2637] (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ek). However, analysis of the discrimination index showed no interaction between DKK1 induction state and time associated with cSVD onset [P\u0026thinsp;=\u0026thinsp;0.6648], with no independent main effects of time associated with cSVD onset [P\u0026thinsp;=\u0026thinsp;0.3578] and DKK1 induction state [P\u0026thinsp;=\u0026thinsp;0.9647], indicating that recognition memory didn\u0026rsquo;t vary over time upon cSVD nor with DKK1 elevated levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003el). Our findings suggest that DKK1 elevated levels promote hyperactive state independently upon cSVD.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.5. cSVD-mediated neuroinflammation is minimally affected by DKK1 induction\u003c/h2\u003e\u003cp\u003eNeuroinflammation underlying ischemic lesions could be detrimental, leading to injury progression when imbalanced, yet also contributes to tissue repair [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Microglia, brain\u0026rsquo;s resident immune cell, and infiltrating myeloid cells are rapidly activated and recruited to the lesion sites upon cSVD associated with microinfarctions, to partake in the inflammatory responses [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. As DKK1 is a potent immunomodulator [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e], we aimed to evaluate the impact of its elevated levels on the neuroinflammatory responses. Immunofluorescence analysis showed that the intensity of IBA-1 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e, expressed in microglia and monocyte-derived macrophages (MDMs), slightly increased in the ipsilateral hemisphere of DKK1/ON mice compared to WT/DOX mice at day 14 after cSVD [P\u0026thinsp;=\u0026thinsp;0.0939] (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). At day 30 after cSVD, following normalization of DKK1 levels, IBA-1 intensity remained unchanged between both groups [P\u0026thinsp;=\u0026thinsp;0.3148] (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Similarly, in the hippocampus, IBA-1 intensity remained unchanged between both groups at 14 days [P\u0026thinsp;=\u0026thinsp;0.1206] and slightly decreased in DKK1/ON\u0026rarr;OFF mice upon normalization of DKK1 levels at 30 days after cSVD [P\u0026thinsp;=\u0026thinsp;0.0878] (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). Next, we assessed the extent of the area covered by reactive IBA-1\u003csup\u003e+\u003c/sup\u003e cells, and we observed a slight increase in the ipsilateral hemisphere of DKK1/ON mice compared to WT/DOX mice at day 14 after cSVD [P\u0026thinsp;=\u0026thinsp;0.1260] (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). No difference in reactive IBA-1\u003csup\u003e+\u003c/sup\u003e cell coverage was observed at day 30 after cSVD upon normalization of DKK1 levels [P\u0026thinsp;=\u0026thinsp;0.2596] (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). Similarly, reactive IBA-1\u003csup\u003e+\u003c/sup\u003e coverage in the hippocampus remained unchanged between both groups at day 14 [P\u0026thinsp;=\u0026thinsp;0.2610] and day 30 [P\u0026thinsp;=\u0026thinsp;0.1535] after cSVD (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee). Our findings indicate that elevated DKK1 levels slightly enhanced microglial reactivity after cSVD, but this effect was completely resolved after normalization of DKK1 expression. Next, we evaluated the overall infiltration of immune cells by immunolabeling CD45, which is also expressed in MDMs \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef\u003cb\u003e)\u003c/b\u003e. Our analysis revealed that CD45 intensity in the ipsilateral hemisphere was similar between both groups at day 14 after cSVD [P\u0026thinsp;=\u0026thinsp;0.2810] (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg). Analysis of the mean CD45 fluorescence signal revealed no significant difference between WT/DOX mice (181.81\u0026plusmn;119.99) and DKK1/ON mice (430.93\u0026plusmn;497.07), but we could appreciate a slight overall increase in DKK1/ON mice at day 14 after cSVD [P\u0026thinsp;=\u0026thinsp;0.2810] (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eh). At day 30 after cSVD, CD45 intensity remained unchanged in WT/DOX\u0026rarr;OFF mice compared to DKK1/ON\u0026rarr;OFF mice [P\u0026thinsp;=\u0026thinsp;0.1331] (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ei). Analysis of the mean CD45 fluorescence signal in the ipsilateral hemisphere outlined an increase in WT/DOX\u0026rarr;OFF mice (46.19\u0026plusmn;43.91) compared to DKK1/ON\u0026rarr;OFF mice (1.07\u0026plusmn;1.09) [P\u0026thinsp;=\u0026thinsp;0.0001] (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ej). This suggests that while the peak of neuroinflammation at day 14 after cSVD is not affected by DKK1, its resolution is accelerated upon normalization of its expression levels. Our findings indicate that neuroinflammatory responses after cSVD are not significantly affected by DKK1 expression levels.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e3.6. DKK1 elevated levels maintain a pro-inflammatory phenotype in monocytes upon cSVD\u003c/h2\u003e\u003cp\u003eCirculating monocytes play an important role in the pathobiology of cSVD associated with microinfarcts [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Murine monocytes can be classified based on Ly6C expression and comprise pro-inflammatory Ly6C\u003csup\u003ehigh\u003c/sup\u003e, patrolling Ly6C\u003csup\u003elow\u003c/sup\u003e and intermediate Ly6C\u003csup\u003einter\u003c/sup\u003e subsets [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Our group has previously demonstrated that monocyte subsets are differentially regulated upon cSVD [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Herein, we used flow cytometry analysis to elucidate the impact of DKK1 elevated circulating levels on the dynamics of different monocyte subsets at baseline, 14 and 30 days after cSVD (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). Prior to cSVD, frequencies in total leukocytes (CD45\u003csup\u003e+\u003c/sup\u003e) [P\u0026thinsp;=\u0026thinsp;0.9430] and total monocytes (CD11b\u003csup\u003e+\u003c/sup\u003eCD115\u003csup\u003e+\u003c/sup\u003e) [P\u0026thinsp;=\u0026thinsp;0.7271], as well as Ly6C\u003csup\u003elow\u003c/sup\u003e [P\u0026thinsp;=\u0026thinsp;0.6821], Ly6C\u003csup\u003einter\u003c/sup\u003e [P\u0026thinsp;=\u0026thinsp;0.7056] and Ly6C\u003csup\u003ehigh\u003c/sup\u003e [P\u0026thinsp;=\u0026thinsp;0.6154] subsets showed no differences between WT/DOX mice and DKK1/ON mice, indicating that DKK1 induction didn\u0026rsquo;t affect basal monocyte distribution (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb-f). Furthermore, frequencies of total leukocytes [P\u0026thinsp;=\u0026thinsp;0.7396] and total monocytes [P\u0026thinsp;=\u0026thinsp;0.1484] between the two groups remained unchanged at day 14 after cSVD (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg, h). Although no significant differences in the frequencies of monocyte subsets at day 14 after cSVD were reported, we observed a slight decrease in the frequency of Ly6C\u003csup\u003elow\u003c/sup\u003e in DKK1/ON mice compared to WT/DOX mice [P\u0026thinsp;=\u0026thinsp;0.1018] (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ei), no difference in the frequency of Ly6C\u003csup\u003einter\u003c/sup\u003e [P\u0026thinsp;=\u0026thinsp;0.7833] (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ej), and a slight increase in the frequency of Ly6C\u003csup\u003ehigh\u003c/sup\u003e [P\u0026thinsp;=\u0026thinsp;0.1180] (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ek) subset in DKK1/ON mice compared to WT/DOX mice. This suggests that circulating monocytes are acquiring a pro-inflammatory phenotype after cSVD when DKK1 circulating levels are elevated. At day 30 after cSVD following normalization of DKK1 levels, no difference in the frequencies of total leukocytes [P\u0026thinsp;=\u0026thinsp;0.6197] and total monocytes [P\u0026thinsp;=\u0026thinsp;0.3638] were noted between both groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003el, m). However, frequency of Ly6C\u003csup\u003elow\u003c/sup\u003e monocytes significantly increased in WT/DOX\u0026rarr;OFF mice compared to DKK1/ON\u0026rarr;OFF mice [P\u0026thinsp;=\u0026thinsp;0.0003] (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003en\u003cb\u003e)\u003c/b\u003e. Frequency of Ly6C\u003csup\u003einter\u003c/sup\u003e monocytes significantly decreased in DKK1/ON\u0026rarr;OFF mice compared to WT/DOX\u0026rarr;OFF mice [P\u0026thinsp;=\u0026thinsp;0.0003] (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eo). Moreover, frequency of Ly6C\u003csup\u003ehigh\u003c/sup\u003e monocytes was strongly reduced in WT/DOX\u0026rarr;OFF mice compared to DKK1/ON\u0026rarr;OFF mice [P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001] (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ep). This indicates that monocytes in WT mice respond to cSVD by increasing the frequency of patrolling Ly6C\u003csup\u003elow\u003c/sup\u003e subset over time (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eq) to partake in neurovascular repair [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. However, DKK1 elevated levels mediate profound changes in the response of monocytes over time, by maintaining an elevated frequency of pro-inflammatory Ly6C\u003csup\u003ehigh\u003c/sup\u003e subset involved in cSVD progression [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eq). Our findings show that DKK1 elevated levels in blood circulation promote an acute-like long lasting pro-inflammatory polarization of monocytes after cSVD.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e3.7. Circulating DKK1 differentially regulates anti- and pro-inflammatory cytokines in serum\u003c/h2\u003e\u003cp\u003eTo further explore the long-term effects of DKK1 induction on systemic inflammatory responses, we quantified the concentration of key cytokines in the serum of WT/DOX\u0026rarr;OFF and DKK1/ON\u0026rarr;OFF mice 30 days after cSVD, following normalization of DKK1 levels. Granulocyte macrophage colony stimulating factor (GM-CSF), a multipotent cytokine that can stimulate proliferation of bone marrow derived macrophages and granulocytes [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e], was detected in 50% of WT/DOX\u0026rarr;OFF mice at a mean concentration of 6.33 pg/mL [5.66\u0026ndash;7.00 pg/mL], while it was undetectable in all DKK1/ON\u0026rarr;OFF mice (\u003cb\u003eTable\u0026nbsp;1\u003c/b\u003e). Anti-inflammatory cytokine interleukin 10 (IL-10) was detected in 50% of both groups with a mean concentration of 2.93 pg/mL [0.36\u0026ndash;5.50 pg/mL] in WT/DOX\u0026rarr;OFF mice and 1.93 pg/mL [1.74\u0026ndash;2.11 pg/mL] in DKK1/ON\u0026rarr;OFF mice, suggesting a slight decrease of IL-10 in the DKK1 transgenic mice (\u003cb\u003eTable\u0026nbsp;1\u003c/b\u003e). Finally, interferon-γ (INF-γ), a pro-inflammatory mediator, was detected in 25% of WT/DOX\u0026rarr;OFF mice at a concentration of 1.63 pg/mL and was detected in 75% of DKK1/ON\u0026rarr;OFF mice with a mean concentration of 1 pg/mL [0.38\u0026ndash;2.14 pg/mL] (\u003cb\u003eTable\u0026nbsp;1\u003c/b\u003e). Our findings suggest that DKK1 high levels in blood circulation at cSVD onset mediate prolonged changes associated with the release of pro-inflammatory cytokines.\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eA better understanding of the mechanisms modulating the pathobiology of cSVD associated with microinfarcts would provide novel insights into the development of effective therapeutic approaches. The current prevention and treatment strategies rely essentially on managing vascular risk factors associated with cSVD, such as hypertension, diabetes, hypercholesterolemia and smoking, through lifestyle modifications and pharmacological interventions [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. DKK1 is an antagonist of the canonical Wnt pathway which plays a key role in maintaining neurovascular integrity and regulating immune functions [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. DKK1 circulating levels are elevated in different pathological conditions, including vascular, immune and metabolic disorders [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. This suggests that DKK1 represents a major factor potentially implicated in aggravating the progression of cSVD, outlining its potential as a prognostic and therapeutic target.\u003c/p\u003e\u003cp\u003eHerein, we show that DKK1 circulating levels potently increase in the days following cSVD, gradually decreasing over time. DKK1 circulating levels have been reported to be elevated in stable cerebrovascular diseases [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Nonetheless, our findings suggest that cSVD associated with microinfarcts triggers DKK1 \u003cem\u003ede novo\u003c/em\u003e release in the blood circulation. As DKK1 elevated levels is commonly reported in various risk factors associated with cSVD, we further elucidated the impact of its conditional induction in modulating the pathobiology of cSVD. This was achieved using innovative tools to mediate DKK1 widespread induction while preserving its endogenous tissue distribution. Our analysis indicates that DKK1 induction in transgenic mice results in greater short-term and long-term weight loss after cSVD. Various reports indicate that weight loss is linked to poor functional outcomes and mortality risk after ischemic stroke and positively correlates with infarct volume and severity, suggesting it could be detrimental as well in the context of cSVD [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. DKK1 widespread expression also tends to exacerbate vascular permeability in the ipsilateral hemisphere, which represents a critical pathological factor in cSVD pathogenesis etiology and clinical progression [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Additionally, we reveal that DKK1 elevated levels are associated with a severe interruption of CBF upon occlusion of the penetrating arterioles. This suggests that elevated DKK1 levels at cSVD onset alter acute vascular reactivity and hinder immediate brain reperfusion, thereby compromising acute neurovascular responses, which underly exacerbation of ischemic injury severity [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe hippocampus is particularly vulnerable to hypoperfusion mediated by CBF impairments associated with cerebrovascular pathologies, including dementia [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. Our group has previously shown that cSVD associated with microinfarcts implicates important neuronal loss in the hippocampus [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Herein, we report that DKK1 elevated levels exacerbate neuronal loss specifically in the CA region. Interestingly, normalization of DKK1 widespread expression after cSVD via withdrawal of doxycycline reduces progressive neuronal loss. This suggests that strategies aiming to block DKK1 action could promote neuroprotection effects in cSVD. It is in line with previous reports indicating that attenuation of DKK1 expression protects neurons in the hippocampus against ischemic insults [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Brain injuries activate neurogenic niches, including DG in the hippocampus, translated by the proliferation, migration, and differentiation of neural stem cells, translating an attempt to replace the lost neurons [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. Evidence is indicating that microinfarcts associated with VaD implicate activation of neurogenic responses [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. Herein, we uncover that DKK1 induction impairs neurogenesis under normal conditions. Notably, DCX\u003csup\u003e+\u003c/sup\u003e neuroblasts in the intact DG upon DKK1 induction exhibit atrophic dendrites. Canonical Wnt pathway regulates dendrite morphogenesis, and its inhibition reduces dendritic length, which could lead to weaker network connectivity [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. Furthermore, activation of canonical Wnt signaling mediated via reduction of DKK1 expression has been demonstrated to enhance neurogenesis in the hippocampus [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Our results indicate that DKK1-induced reduction of neurogenesis, before and after cSVD, impairs endogenous neurorestorative mechanisms. cSVD is associated with various clinical manifestations depending upon the affected brain region [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. We show that anxiety-like behaviors in mice are increased upon cSVD and that DKK1 elevated levels are associated with the emergence of a hyperactive behavior. Hyperactivity or increased spontaneous activity in rodents, presumably translating an agitated state, are often observed in VaD as well as at the early stages of AD [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eNeuroinflammation plays an important role in the pathogenesis of cSVD [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. DKK1 acts as an immunomodulator under various pathological conditions [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. Herein, we aimed to investigate the impact of DKK1 elevated levels on immune responses in cSVD associated with microinfarcts. Microglial cell activation plays a multifaceted role in brain injuries associated with infarctions by either contributing to tissue repair via removal of debris and release of trophic factors, or exacerbating damage via excessive release of pro-inflammatory mediators [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. In our study, we show that DKK1 elevated levels enhance microglial reactivity after cSVD, which is attenuated over time upon normalization of its expression levels. Infiltrating myeloid cells represent as well an important component of neuroinflammation after injury [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Our analysis suggests that peripheral immune cell infiltration slightly increases with DKK1 elevated levels, an effect that is reversed upon normalization of its expression levels. This suggest the DKK1 levels may contribute to the establishment of an inflammatory microenvironment that impedes repair. In addition to resident microglia, ischemic insults trigger the recruitment of monocytes that differentiate into MDMs [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]. Our group previously showed that monocyte subsets are differentially modulated after cSVD, affecting injury progression [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Herein, we reveal that circulating Ly6C\u003csup\u003ehigh\u003c/sup\u003e inflammatory monocytes progressively transition into Ly6C\u003csup\u003elow\u003c/sup\u003e monocytes, which play a critical role in preserving neurovascular functions upon cSVD [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. However, DKK1 elevated levels promote a persisting pro-inflammatory phenotype in circulating monocytes after cSVD despite normalization of its expression levels. Interestingly, Ly6C\u003csup\u003ehigh\u003c/sup\u003e inflammatory monocytes are characterized by an increased capacity of cytokine production and are linked to cSVD progression [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. This suggests that DKK1 elevated levels promote the accumulation of pro-inflammatory monocytes in blood circulation, possibly contributing to exacerbation of systemic inflammation and subsequently injury progression upon cSVD.\u003c/p\u003e\u003cp\u003eDifferent studies outlined an association between circulating inflammatory cytokines with cSVD onset and progression [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]. Although GM-CSF is mainly recognized as a pro-inflammatory cytokine, evidence suggests it could act as an anti-inflammatory cytokine depending on the context [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e]. Herein, we show that GM-CSF is undetectable in inducible DKK1 transgenic mice after cSVD upon DKK1 normalization, correlating with present systemic inflammation as indicated by the increased frequency of pro-inflammatory monocytes. Nonetheless, GM-CSF is detected in 50% of WT mice in which an increased frequency of Ly6C\u003csup\u003elow\u003c/sup\u003e monocyte is reported. This outlines a contribution to resolution of inflammation under normal conditions after cSVD. IL-10 is detectable in 50% of both groups, but at slightly lower levels in inducible DKK1 transgenic mice upon DKK1 normalization. IL-10 is endogenously produced by immune cells to limit the inflammatory reaction induced by ischemic insults and its administration has been shown to provide neuroprotection in experimental stroke [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e]. INF-γ is a pro-inflammatory mediator that contributes to inflammatory and thrombogenic responses in ischemic stroke [\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e]. We detected INF-γ in 75% of inducible DKK1 transgenic mice and in only 25% of WT mice. Herein, we uncover that DKK1 elevated levels at cSVD onset is sufficient to mediate profound changes in the systemic inflammatory responses that persist over time despite normalization of its circulating levels, an aspect that should be taken into consideration.\u003c/p\u003e\u003cp\u003eOur study provides new insights into the role of DKK1 in cSVD pathobiology via deregulation of immune-neurovascular functions. Our findings suggest that DKK1 circulating levels would provide valuable information about brain injury progression associated with cSVD and outline its potential as a promising target for the development of clinically relevant disease-modifying therapies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e5.\u003c/strong\u003e \u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank Natija Aldib for helping in the LSCI experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6. Author contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors contributed as follows; Esther Trudel: Experimentation, data interpretation, figure preparation, drafting and editing. Anne-Sophie Allain: Experimentation, and editing. Ayman ElAli; Conceptualization, data interpretation, drafting and editing.\u0026nbsp;All authors approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e7. Funding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work is supported by grants from the Heart and Stroke Foundation of Canada (HSFC) (#G-23-0035047), and the Canadian Institutes of Health Research (CIHR) (#169062; #186148) (all to Ayman ElAli). Ayman ElAli holds a Tier 2 Canada Research Chair in molecular and cellular neurovascular interactions. Esther Trudel is the recipient of the scholarship of excellence Didier Mouginot of the \u003cem\u003eFondation du CHU de Qu\u0026eacute;bec \u0026ndash; Desjardins\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e8. Data and materials availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll other data are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e9.1. Ethics approval:\u0026nbsp;\u003c/strong\u003eThe experimental protocol was conducted in accordance with the ethical policies and procedures approved by the committee of animal protection of Universit\u0026eacute; Laval (Comit\u0026eacute; de Protection des Animaux de l\u0026apos;Universit\u0026eacute; Laval (CPAUL)-3; Approval # 22-1136). Handling of animals adhered to the principles outlined in the Canadian Council on Animal Care (CCAC) guidelines. Animal studies were reported according to ARRIVE 2.0 guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e9.2.\u003c/strong\u003e \u003cstrong\u003eConsent to participate:\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e9.3. Competing interests:\u003c/strong\u003e The authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e9.4. Clinical Trial Number:\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e9.5. Consent for publication:\u003c/strong\u003e Not applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eChojdak-Łukasiewicz J, Dziadkowiak E, Zimny A, Paradowski B. Cerebral small vessel disease: A review. Adv Clin Exp Med. 2021;30(3):349\u0026ndash;56. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.17219/acem/131216\u003c/span\u003e\u003cspan address=\"10.17219/acem/131216\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ede Leeuw FE, de Groot JC, Achten E, et al. Prevalence of cerebral white matter lesions in elderly people: a population based magnetic resonance imaging study. The Rotterdam Scan Study. J Neurol Neurosurg Psychiatry. 2001;70(1):9\u0026ndash;14. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1136/jnnp.70.1.9\u003c/span\u003e\u003cspan address=\"10.1136/jnnp.70.1.9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePetty GW, Brown RD, Whisnant JP, Sicks JD, O\u0026rsquo;Fallon WM, Wiebers DO. Ischemic stroke subtypes: a population-based study of functional outcome, survival, and recurrence. Stroke. 2000;31(5):1062\u0026ndash;8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1161/01.str.31.5.1062\u003c/span\u003e\u003cspan address=\"10.1161/01.str.31.5.1062\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRensma SP, van Sloten TT, Launer LJ, Stehouwer CDA. Cerebral small vessel disease and risk of incident stroke, dementia and depression, and all-cause mortality: A systematic review and meta-analysis. Neurosci Biobehav Rev. 2018;90:164\u0026ndash;73. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.neubiorev.2018.04.003\u003c/span\u003e\u003cspan address=\"10.1016/j.neubiorev.2018.04.003\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWardlaw JM, Smith EE, Biessels GJ, et al. Neuroimaging standards for research into small vessel disease and its contribution to ageing and neurodegeneration. Lancet Neurol. 2013;12(8):822\u0026ndash;38. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/S1474-4422(13)70124-8\u003c/span\u003e\u003cspan address=\"10.1016/S1474-4422(13)70124-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePantoni L. Cerebral small vessel disease: from pathogenesis and clinical characteristics to therapeutic challenges. Lancet Neurol. 2010;9(7):689\u0026ndash;701. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/S1474-4422(10)70104-6\u003c/span\u003e\u003cspan address=\"10.1016/S1474-4422(10)70104-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWardlaw JM, Smith C, Dichgans M. Small vessel disease: mechanisms and clinical implications. Lancet Neurol. 2019;18(7):684\u0026ndash;96. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/S1474-4422(19)30079-1\u003c/span\u003e\u003cspan address=\"10.1016/S1474-4422(19)30079-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGorelick PB, Scuteri A, Black SE, et al. Vascular contributions to cognitive impairment and dementia: a statement for healthcare professionals from the american heart association/american stroke association. Stroke. 2011;42(9):2672\u0026ndash;713. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1161/STR.0b013e3182299496\u003c/span\u003e\u003cspan address=\"10.1161/STR.0b013e3182299496\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKhan U, Porteous L, Hassan A, Markus HS. Risk factor profile of cerebral small vessel disease and its subtypes. J Neurol Neurosurg Psychiatry. 2007;78(7):702\u0026ndash;6. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1136/jnnp.2006.103549\u003c/span\u003e\u003cspan address=\"10.1136/jnnp.2006.103549\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi Q, Yang Y, Reis C, et al. Cerebral Small Vessel Disease. Cell Transpl. 2018;27(12):1711\u0026ndash;22. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1177/0963689718795148\u003c/span\u003e\u003cspan address=\"10.1177/0963689718795148\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSilasi G, She J, Boyd JD, Xue S, Murphy TH. A mouse model of small-vessel disease that produces brain-wide-identified microocclusions and regionally selective neuronal injury. J Cereb Blood Flow Metab. 2015;35(5):734\u0026ndash;8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/jcbfm.2015.8\u003c/span\u003e\u003cspan address=\"10.1038/jcbfm.2015.8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBalbi M, Vanni MP, Vega MJ, et al. Longitudinal monitoring of mesoscopic cortical activity in a mouse model of microinfarcts reveals dissociations with behavioral and motor function. J Cereb Blood Flow Metab. 2019;39(8):1486\u0026ndash;500. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1177/0271678X18763428\u003c/span\u003e\u003cspan address=\"10.1177/0271678X18763428\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHermann DM, ElAli A. The Abluminal Endothelial Membrane in Neurovascular Remodeling in Health and Disease. Sci Signal. 2012;5(236). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1126/scisignal.2002886\u003c/span\u003e\u003cspan address=\"10.1126/scisignal.2002886\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMenet R, Lecordier S, ElAli A. Wnt Pathway: An Emerging Player in Vascular and Traumatic Mediated Brain Injuries. Front Physiol. 2020;11:565667. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fphys.2020.565667\u003c/span\u003e\u003cspan address=\"10.3389/fphys.2020.565667\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMarchetti B, Pluchino S. Wnt your brain be inflamed? Yes, it Wnt! Trends Mol Med. 2013;19(3):144\u0026ndash;56. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.molmed.2012.12.001\u003c/span\u003e\u003cspan address=\"10.1016/j.molmed.2012.12.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMastroiacovo F, Busceti CL, Biagioni F, et al. Induction of the Wnt antagonist, Dickkopf-1, contributes to the development of neuronal death in models of brain focal ischemia. J Cereb Blood Flow Metab. 2009;29(2):264\u0026ndash;76. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/jcbfm.2008.111\u003c/span\u003e\u003cspan address=\"10.1038/jcbfm.2008.111\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang QG, Wang R, Khan M, Mahesh V, Brann DW. Role of Dickkopf-1, an antagonist of the Wnt/beta-catenin signaling pathway, in estrogen-induced neuroprotection and attenuation of tau phosphorylation. J Neurosci. 2008;28(34):8430\u0026ndash;41. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1523/JNEUROSCI.2752-08.2008\u003c/span\u003e\u003cspan address=\"10.1523/JNEUROSCI.2752-08.2008\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMenet R, Bourassa P, Calon F, ElAli A. Dickkopf-related protein-1 inhibition attenuates amyloid-beta pathology associated to Alzheimer\u0026rsquo;s disease. Neurochem Int. 2020;141:104881. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.neuint.2020.104881\u003c/span\u003e\u003cspan address=\"10.1016/j.neuint.2020.104881\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHimburg HA, Doan PL, Quarmyne M, et al. Dickkopf-1 promotes hematopoietic regeneration via direct and niche-mediated mechanisms. Nat Med. 2017;23(1):91\u0026ndash;9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/nm.4251\u003c/span\u003e\u003cspan address=\"10.1038/nm.4251\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSeifert-Held T, Pekar T, Gattringer T, et al. Circulating Dickkopf-1 in acute ischemic stroke and clinically stable cerebrovascular disease. Atherosclerosis. 2011;218(1):233\u0026ndash;7. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.atherosclerosis.2011.05.015\u003c/span\u003e\u003cspan address=\"10.1016/j.atherosclerosis.2011.05.015\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHe XW, Wang E, Bao YY, et al. High serum levels of sclerostin and Dickkopf-1 are associated with acute ischaemic stroke. Atherosclerosis. 2016;253:22\u0026ndash;8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.atherosclerosis.2016.08.003\u003c/span\u003e\u003cspan address=\"10.1016/j.atherosclerosis.2016.08.003\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhu Z, Guo D, Zhong C, et al. Serum Dkk-1 (Dickkopf-1) Is a Potential Biomarker in the Prediction of Clinical Outcomes Among Patients With Acute Ischemic Stroke. Arterioscler Thromb Vasc Biol. 2019;39(2):285\u0026ndash;93. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1161/ATVBAHA.118.311960\u003c/span\u003e\u003cspan address=\"10.1161/ATVBAHA.118.311960\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang K, Zhu Z, Shi M, et al. Serum Dickkopf-1 levels and poststroke depression in ischemic stroke patients. J Affect Disord. 2022;310:337\u0026ndash;42. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.jad.2022.05.040\u003c/span\u003e\u003cspan address=\"10.1016/j.jad.2022.05.040\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAbraham HMA, Wolfson L, Moscufo N, Guttmann CRG, Kaplan RF, White WB. Cardiovascular risk factors and small vessel disease of the brain: Blood pressure, white matter lesions, and functional decline in older persons. J Cereb Blood Flow Metab. 2016;36(1):132\u0026ndash;42. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/jcbfm.2015.121\u003c/span\u003e\u003cspan address=\"10.1038/jcbfm.2015.121\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCappuccio I, Calderone A, Busceti CL, et al. Induction of Dickkopf-1, a Negative Modulator of the Wnt Pathway, Is Required for the Development of Ischemic Neuronal Death. J Neurosci. 2005;25(10):2647\u0026ndash;57. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1523/JNEUROSCI.5230-04.2005\u003c/span\u003e\u003cspan address=\"10.1523/JNEUROSCI.5230-04.2005\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eXu H, Ding Z, Chen J, et al. Correlation between serum Dickkopf-1 (DKK1) levels and coronary artery stenosis. Nutr Metabolism Cardiovasc Dis. 2023;33(1):168\u0026ndash;76. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.numecd.2022.10.007\u003c/span\u003e\u003cspan address=\"10.1016/j.numecd.2022.10.007\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLattanzio S, Santilli F, Liani R, et al. Circulating Dickkopf-1 in Diabetes Mellitus: Association With Platelet Activation and Effects of Improved Metabolic Control and Low‐Dose Aspirin. J Am Heart Assoc. 2014;3(4):e001000. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1161/JAHA.114.001000\u003c/span\u003e\u003cspan address=\"10.1161/JAHA.114.001000\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLecordier S, Manrique-Castano D, El Moghrabi Y, ElAli A. Neurovascular Alterations in Vascular Dementia: Emphasis on Risk Factors. Front Aging Neurosci. 2021;13:727590. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fnagi.2021.727590\u003c/span\u003e\u003cspan address=\"10.3389/fnagi.2021.727590\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVolckaert T, Dill E, Campbell A, et al. Parabronchial smooth muscle constitutes an airway epithelial stem cell niche in the mouse lung after injury. J Clin Invest. 2011;121(11):4409\u0026ndash;19. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1172/JCI58097\u003c/span\u003e\u003cspan address=\"10.1172/JCI58097\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCvetkovic B, Yang B, Williamson RA, Sigmund CD. Appropriate tissue- and cell-specific expression of a single copy human angiotensinogen transgene specifically targeted upstream of the HPRT locus by homologous recombination. J Biol Chem. 2000;275(2):1073\u0026ndash;8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1074/jbc.275.2.1073\u003c/span\u003e\u003cspan address=\"10.1074/jbc.275.2.1073\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHeaney JD, Rettew AN, Bronson SK. Tissue-specific expression of a BAC transgene targeted to the Hprt locus in mouse embryonic stem cells. Genomics. 2004;83(6):1072\u0026ndash;82. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.ygeno.2003.12.015\u003c/span\u003e\u003cspan address=\"10.1016/j.ygeno.2003.12.015\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSeibenhener ML, Wooten MC. Use of the Open Field Maze to measure locomotor and anxiety-like behavior in mice. J Vis Exp. 2015;96e52434. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3791/52434\u003c/span\u003e\u003cspan address=\"10.3791/52434\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCrawley J, Goodwin FK. Preliminary report of a simple animal behavior model for the anxiolytic effects of benzodiazepines. Pharmacol Biochem Behav. 1980;13(2):167\u0026ndash;70. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/0091-3057(80)90067-2\u003c/span\u003e\u003cspan address=\"10.1016/0091-3057(80)90067-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBourin M, Hasco\u0026euml;t M. The mouse light/dark box test. Eur J Pharmacol. 2003;463(1):55\u0026ndash;65. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/S0014-2999(03)01274-3\u003c/span\u003e\u003cspan address=\"10.1016/S0014-2999(03)01274-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAl-Onaizi MA, Th\u0026eacute;riault P, Lecordier S, Prefontaine P, Rivest S, ElAli A. Early monocyte modulation by the non-erythropoietic peptide ARA 290 decelerates AD-like pathology progression. Brain Behav Immun. 2022;99:363\u0026ndash;82. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.bbi.2021.07.016\u003c/span\u003e\u003cspan address=\"10.1016/j.bbi.2021.07.016\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTh\u0026eacute;riault P, ElAli A, Rivest S. High fat diet exacerbates Alzheimer\u0026rsquo;s disease-related pathology in APPswe/PS1 mice. Oncotarget. 2016;7(42):67808\u0026ndash;27. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.18632/oncotarget.12179\u003c/span\u003e\u003cspan address=\"10.18632/oncotarget.12179\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBernard M, Menet R, Lecordier S, ElAli A. Endothelial PDGF-D contributes to neurovascular protection after ischemic stroke by rescuing pericyte functions. Cell Mol Life Sci. 2024;81(1):225. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s00018-024-05244-w\u003c/span\u003e\u003cspan address=\"10.1007/s00018-024-05244-w\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJean LeBlanc N, Menet R, Picard K, Parent G, Tremblay M\u0026Egrave;, ElAli A. Canonical Wnt Pathway Maintains Blood-Brain Barrier Integrity upon Ischemic Stroke and Its Activation Ameliorates Tissue Plasminogen Activator Therapy. Mol Neurobiol. 2019;56(9):6521\u0026ndash;38. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s12035-019-1539-9\u003c/span\u003e\u003cspan address=\"10.1007/s12035-019-1539-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTh\u0026eacute;riault P, Le B\u0026eacute;hot A, ElAli A, Rivest S. Sub-acute systemic erythropoietin administration reduces ischemic brain injury in an age-dependent manner. Oncotarget. 2016;7(24):35552\u0026ndash;61. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.18632/oncotarget.9652\u003c/span\u003e\u003cspan address=\"10.18632/oncotarget.9652\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYang Q, Wei X, Deng B, et al. Cerebral small vessel disease alters neurovascular unit regulation of microcirculation integrity involved in vascular cognitive impairment. Neurobiol Dis. 2022;170:105750. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.nbd.2022.105750\u003c/span\u003e\u003cspan address=\"10.1016/j.nbd.2022.105750\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLecordier S, Pons V, Rivest S, ElAli A. Multifocal Cerebral Microinfarcts Modulate Early Alzheimer\u0026rsquo;s Disease Pathology in a Sex-Dependent Manner. Front Immunol. 2022;12:813536. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fimmu.2021.813536\u003c/span\u003e\u003cspan address=\"10.3389/fimmu.2021.813536\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLecordier S, Menet R, Allain AS, ElAli A. Non-classical monocytes promote neurovascular repair in cerebral small vessel disease associated with microinfarctions via CX3CR1. J Cereb Blood Flow Metab. 2023;43(11):1873\u0026ndash;90. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1177/0271678X231183742\u003c/span\u003e\u003cspan address=\"10.1177/0271678X231183742\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSeib DRM, Corsini NS, Ellwanger K, et al. Loss of Dickkopf-1 restores neurogenesis in old age and counteracts cognitive decline. Cell Stem Cell. 2013;12(2):204\u0026ndash;14. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.stem.2012.11.010\u003c/span\u003e\u003cspan address=\"10.1016/j.stem.2012.11.010\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eClancy U, Gilmartin D, Jochems ACC, Knox L, Doubal FN, Wardlaw JM. Neuropsychiatric symptoms associated with cerebral small vessel disease: a systematic review and meta-analysis. Lancet Psychiatry. 2021;8(3):225\u0026ndash;36. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/S2215-0366(20)30431-4\u003c/span\u003e\u003cspan address=\"10.1016/S2215-0366(20)30431-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang J, Zhu G, Huang L, et al. Morphine administration induces change in anxiety-related behavior via Wnt/β-catenin signaling. Neurosci Lett. 2017;639:199\u0026ndash;206. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.neulet.2017.01.005\u003c/span\u003e\u003cspan address=\"10.1016/j.neulet.2017.01.005\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMatrisciano F, Busceti CL, Bucci D, et al. Induction of the Wnt antagonist Dickkopf-1 is involved in stress-induced hippocampal damage. PLoS ONE. 2011;6(1):e16447. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1371/journal.pone.0016447\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0016447\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJayaraj RL, Azimullah S, Beiram R, Jalal FY, Rosenberg GA. Neuroinflammation: friend and foe for ischemic stroke. J Neuroinflammation. 2019;16(1):142. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s12974-019-1516-2\u003c/span\u003e\u003cspan address=\"10.1186/s12974-019-1516-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCandelario-Jalil E, Dijkhuizen RM, Magnus T, Neuroinflammation. Stroke, Blood-Brain Barrier Dysfunction, and Imaging Modalities. Stroke. 2022;53(5):1473\u0026ndash;86. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1161/STROKEAHA.122.036946\u003c/span\u003e\u003cspan address=\"10.1161/STROKEAHA.122.036946\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePark MH, Sung EA, Sell M, Chae WJ. Dickkopf1: An Immunomodulator in Tissue Injury, Inflammation, and Repair. ImmunoHorizons. 2021;5(11):898\u0026ndash;908. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.4049/immunohorizons.2100015\u003c/span\u003e\u003cspan address=\"10.4049/immunohorizons.2100015\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNoz MP, Telgte A, ter, Wiegertjes K, et al. Pro-inflammatory Monocyte Phenotype During Acute Progression of Cerebral Small Vessel Disease. Front Cardiovasc Med. 2021;8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fcvm.2021.639361\u003c/span\u003e\u003cspan address=\"10.3389/fcvm.2021.639361\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eElAli A, Jean LeBlanc N. The Role of Monocytes in Ischemic Stroke Pathobiology: New Avenues to Explore. Front Aging Neurosci. 2016;8:29. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fnagi.2016.00029\u003c/span\u003e\u003cspan address=\"10.3389/fnagi.2016.00029\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNoz MP, Ter Telgte A, Wiegertjes K, et al. Pro-inflammatory Monocyte Phenotype During Acute Progression of Cerebral Small Vessel Disease. Front Cardiovasc Med. 2021;8:639361. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fcvm.2021.639361\u003c/span\u003e\u003cspan address=\"10.3389/fcvm.2021.639361\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLotfi N, Zhang GX, Esmaeil N, Rostami A. Evaluation of the effect of GM-CSF blocking on the phenotype and function of human monocytes. Sci Rep. 2020;10(1):1567. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41598-020-58131-2\u003c/span\u003e\u003cspan address=\"10.1038/s41598-020-58131-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWardlaw JM, Allerhand M, Doubal FN, et al. Vascular risk factors, large-artery atheroma, and brain white matter hyperintensities. Neurology. 2014;82(15):1331\u0026ndash;8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1212/WNL.0000000000000312\u003c/span\u003e\u003cspan address=\"10.1212/WNL.0000000000000312\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCannistraro RJ, Badi M, Eidelman BH, Dickson DW, Middlebrooks EH, Meschia JF. CNS small vessel disease. Neurology. 2019;92(24):1146\u0026ndash;56. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1212/WNL.0000000000007654\u003c/span\u003e\u003cspan address=\"10.1212/WNL.0000000000007654\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eUeland T, Otterdal K, Lekva T, et al. Dickkopf-1 enhances inflammatory interaction between platelets and endothelial cells and shows increased expression in atherosclerosis. Arterioscler Thromb Vasc Biol. 2009;29(8):1228\u0026ndash;34. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1161/ATVBAHA.109.189761\u003c/span\u003e\u003cspan address=\"10.1161/ATVBAHA.109.189761\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCai L, Geng X, Hussain M, et al. Weight loss: indication of brain damage and effect of combined normobaric oxygen and ethanol therapy after stroke. Neurol Res. 2015;37(5):441\u0026ndash;6. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1179/1743132815Y.0000000033\u003c/span\u003e\u003cspan address=\"10.1179/1743132815Y.0000000033\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKim Y, Kim CK, Jung S, Ko SB, Lee SH, Yoon BW. Prognostic importance of weight change on short-term functional outcome in acute ischemic stroke. Int J Stroke. 2015;10(Suppl A):62\u0026ndash;8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/ijs.12554\u003c/span\u003e\u003cspan address=\"10.1111/ijs.12554\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWong SM, Jansen JFA, Zhang CE, et al. Blood-brain barrier impairment and hypoperfusion are linked in cerebral small vessel disease. Neurology. 2019;92(15):e1669\u0026ndash;77. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1212/WNL.0000000000007263\u003c/span\u003e\u003cspan address=\"10.1212/WNL.0000000000007263\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSchreiber S, Bueche CZ, Garz C, Braun H. Blood brain barrier breakdown as the starting point of cerebral small vessel disease? - New insights from a rat model. Experimental Translational Stroke Med. 2013;5(1):4. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/2040-7378-5-4\u003c/span\u003e\u003cspan address=\"10.1186/2040-7378-5-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKUNZ A, IADECOLA C. Cerebral vascular dysregulation in the ischemic brain. Handb Clin Neurol. 2009;92:283\u0026ndash;305. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/S0072-9752(08)01914-3\u003c/span\u003e\u003cspan address=\"10.1016/S0072-9752(08)01914-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJohnson AC. Hippocampal Vascular Supply and Its Role in Vascular Cognitive Impairment. Stroke. 2023;54(3):673\u0026ndash;85. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1161/STROKEAHA.122.038263\u003c/span\u003e\u003cspan address=\"10.1161/STROKEAHA.122.038263\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eArvidsson A, Collin T, Kirik D, Kokaia Z, Lindvall O. Neuronal replacement from endogenous precursors in the adult brain after stroke. Nat Med. 2002;8(9):963\u0026ndash;70. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/nm747\u003c/span\u003e\u003cspan address=\"10.1038/nm747\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNakatomi H, Kuriu T, Okabe S, et al. Regeneration of hippocampal pyramidal neurons after ischemic brain injury by recruitment of endogenous neural progenitors. Cell. 2002;110(4):429\u0026ndash;41. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/s0092-8674(02)00862-0\u003c/span\u003e\u003cspan address=\"10.1016/s0092-8674(02)00862-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMacas J, Nern C, Plate KH, Momma S. Increased generation of neuronal progenitors after ischemic injury in the aged adult human forebrain. J Neurosci. 2006;26(50):13114\u0026ndash;9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1523/JNEUROSCI.4667-06.2006\u003c/span\u003e\u003cspan address=\"10.1523/JNEUROSCI.4667-06.2006\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEkonomou A, Ballard CG, Pathmanaban ON, et al. Increased neural progenitors in vascular dementia. Neurobiol Aging. 2011;32(12):2152\u0026ndash;61. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.neurobiolaging.2010.01.007\u003c/span\u003e\u003cspan address=\"10.1016/j.neurobiolaging.2010.01.007\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCiani L, Salinas PC. WNTs in the vertebrate nervous system: from patterning to neuronal connectivity. Nat Rev Neurosci. 2005;6(5):351\u0026ndash;62. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/nrn1665\u003c/span\u003e\u003cspan address=\"10.1038/nrn1665\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHattori Y, Enmi Jichiro, Kitamura A, et al. A novel mouse model of subcortical infarcts with dementia. J Neurosci. 2015;35(9):3915\u0026ndash;28. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1523/JNEUROSCI.3970-14.2015\u003c/span\u003e\u003cspan address=\"10.1523/JNEUROSCI.3970-14.2015\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang T, Chen Y, Zou Y, et al. Locomotor Hyperactivity in the Early-Stage Alzheimer\u0026rsquo;s Disease-like Pathology of APP/PS1 Mice: Associated with Impaired Polarization of Astrocyte Aquaporin 4. Aging Dis. 2022;13(5):1504\u0026ndash;22. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.14336/AD.2022.0219\u003c/span\u003e\u003cspan address=\"10.14336/AD.2022.0219\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLow A, Mak E, Rowe JB, Markus HS, O\u0026rsquo;Brien JT. Inflammation and cerebral small vessel disease: A systematic review. Ageing Res Rev. 2019;53:100916. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.arr.2019.100916\u003c/span\u003e\u003cspan address=\"10.1016/j.arr.2019.100916\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNeumann H, Kotter MR, Franklin RJM. Debris clearance by microglia: an essential link between degeneration and regeneration. Brain. 2009;132(Pt 2):288\u0026ndash;95. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/brain/awn109\u003c/span\u003e\u003cspan address=\"10.1093/brain/awn109\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGuruswamy R, ElAli A. Complex Roles of Microglial Cells in Ischemic Stroke Pathobiology: New Insights and Future Directions. Int J Mol Sci. 2017;18(3):496. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/ijms18030496\u003c/span\u003e\u003cspan address=\"10.3390/ijms18030496\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWerner Y, Mass E, Ashok Kumar P, et al. Cxcr4 distinguishes HSC-derived monocytes from microglia and reveals monocyte immune responses to experimental stroke. Nat Neurosci. 2020;23(3):351\u0026ndash;62. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41593-020-0585-y\u003c/span\u003e\u003cspan address=\"10.1038/s41593-020-0585-y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYang Y, Yao Z, Huo L. Causal effect of circulating cytokines on MRI markers of cerebral small vessel disease: A mendelian randomization study. Cytokine. 2024;182:156713. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.cyto.2024.156713\u003c/span\u003e\u003cspan address=\"10.1016/j.cyto.2024.156713\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBhattacharya P, Budnick I, Singh M, et al. Dual Role of GM-CSF as a Pro-Inflammatory and a Regulatory Cytokine: Implications for Immune Therapy. J Interferon Cytokine Res. 2015;35(8):585\u0026ndash;99. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1089/jir.2014.0149\u003c/span\u003e\u003cspan address=\"10.1089/jir.2014.0149\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSpera PA, Ellison JA, Feuerstein GZ, Barone FC. IL-10 reduces rat brain injury following focal stroke. Neurosci Lett. 1998;251(3):189\u0026ndash;92. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/s0304-3940(98)00537-0\u003c/span\u003e\u003cspan address=\"10.1016/s0304-3940(98)00537-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYilmaz G, Arumugam TV, Stokes KY, Granger DN. Role of T Lymphocytes and Interferon-γ in Ischemic Stroke. Circulation. 2006;113(17):2105\u0026ndash;12. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1161/CIRCULATIONAHA.105.593046\u003c/span\u003e\u003cspan address=\"10.1161/CIRCULATIONAHA.105.593046\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cimg 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\"\u003e\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"discover-neuroscience","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ndev","sideBox":"Learn more about [Neural Development](http://neuraldevelopment.biomedcentral.com/)","snPcode":"13064","submissionUrl":"https://submission.nature.com/new-submission/13064/3","title":"Discover Neuroscience","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Dickkopf-1, cerebral small vessel disease (cSVD), microinfarcts, neurovascular functions, monocytes, inflammation","lastPublishedDoi":"10.21203/rs.3.rs-7622603/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7622603/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCerebral small vessel disease (cSVD) associated with the occlusion of small penetrating arterioles leading to multifocal microinfarcts, constitutes common microangiopathies affecting brain functions. Dickkopf-1 (DKK1) is a potent inhibitor of the canonical Wnt pathway, which plays key roles in regulating neurovascular and immune functions. DKK1 levels are reported to be increased in cerebrovascular diseases, correlating with poor prognosis. Herein, we aimed to investigate the contribution of DKK1 elevated levels to the pathobiology of cSVD associated with microinfarcts. This was achieved using a transgenic mouse model that enables a conditional tissue-specific induction of DKK1. cSVD was induced via intravascular injection of micro-emboli to occlude penetrating arterioles. Our results indicate that the circulating levels of endogenous DKK1 are increased after cSVD and remain steadily elevated before progressively returning to basal levels. DKK1 conditional induction with respect to its temporal regulation after cSVD exacerbates vascular permeability and alters immediate cerebrovascular reactivity, outlining deregulation of the neurovascular functions. Furthermore, DKK1 induction promotes progressive neuronal loss and impairs neurogenesis after cSVD. We show that DKK1 elevated levels are associated with attenuation of myeloid cell recruitment to the lesion sites, accompanied with a persistent polarization of monocytes in the blood circulation towards a pro-inflammatory phenotype. Notably, we reveal that DKK1 elevated levels for a prolonged period after cSVD mediates pathological changes that remain persistent despite delayed normalization of its expression. Our study suggests that circulating DKK1 decisively affects the pathobiology of cSVD associated with microinfarcts, outlining its potential as a prognostic marker and a therapeutic target.\u003c/p\u003e","manuscriptTitle":"Dickkopf-1 accentuates neuronal damage upon multifocal cerebral microinfarctions by impairing vascular and immune responses","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-29 09:47:55","doi":"10.21203/rs.3.rs-7622603/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-20T08:45:36+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-30T22:14:59+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-27T18:38:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"33649473575179929724112722585627950037","date":"2025-10-20T11:28:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"74548683137309489887754449130968000952","date":"2025-10-19T06:28:10+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-16T10:08:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"48022308770104376244886690537092242822","date":"2025-10-15T11:43:20+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-15T11:25:39+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-03T05:29:02+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-24T15:14:14+00:00","index":"","fulltext":""},{"type":"submitted","content":"Discover Neuroscience","date":"2025-09-24T15:07:12+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"discover-neuroscience","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ndev","sideBox":"Learn more about [Neural Development](http://neuraldevelopment.biomedcentral.com/)","snPcode":"13064","submissionUrl":"https://submission.nature.com/new-submission/13064/3","title":"Discover Neuroscience","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"acb38209-73fe-4215-845f-0c550c16bb28","owner":[],"postedDate":"October 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-01-12T16:10:19+00:00","versionOfRecord":{"articleIdentity":"rs-7622603","link":"https://doi.org/10.1186/s13064-025-00230-9","journal":{"identity":"discover-neuroscience","isVorOnly":false,"title":"Discover Neuroscience"},"publishedOn":"2026-01-06 15:59:15","publishedOnDateReadable":"January 6th, 2026"},"versionCreatedAt":"2025-10-29 09:47:55","video":"","vorDoi":"10.1186/s13064-025-00230-9","vorDoiUrl":"https://doi.org/10.1186/s13064-025-00230-9","workflowStages":[]},"version":"v1","identity":"rs-7622603","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7622603","identity":"rs-7622603","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}