Tweak Promotes Neovascularization And Brain Damage Reduction In A Rat Model Of Intracerebral Hemorrhage

Tweak Promotes Neovascularization And Brain Damage Reduction In A Rat Model Of Intracerebral Hemorrhage | 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 Tweak Promotes Neovascularization And Brain Damage Reduction In A Rat Model Of Intracerebral Hemorrhage Daniel Romaus-Sanjurjo, Esteban López-Arias, Cristina Rodríguez, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5796126/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Non-traumatic intracerebral hemorrhage (ICH) is one of the most devastating and disabling forms of stroke; however, there are no effective pharmacological therapies following the insult. Angiogenesis appears as a key step to overcome the damage and promote functional recovery. In this context, endothelial progenitor cells (EPCs) mobilization promotes neovascularization which has been linked to beneficial outcomes following both ischemic and hemorrhagic stroke. The TNF-like weak inducer of apoptosis (TWEAK), binding to its receptor Fn14, has been suggested as an inducer of EPCs differentiation, viability and migration to the injury site in a model of myocardial infarction. Here, we have performed a proof-of-concept preclinical study in a rat model of ICH where we report that a 50 µg/kg dose of rat recombinant TWEAK (rTWEAK) promotes EPCs mobilization, as soon as 72 h post-injury, brain neovascularization, and, importantly, long-term hematoma reduction and functional recovery. In contrast, a higher dose of 150 µg/kg blocked those beneficial outcomes. Therefore, a low-dose of rTWEAK treatment promotes neovascularization and reduces brain damage in a rat model of ICH. Further clinical studies will be needed to demonstrate if rTWEAK could represent a new strategy to promote recovery following ICH. endothelial progenitor cells intracerebral hemorrhage neovascularization TWEAK Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION Non-traumatic intracerebral hemorrhage (ICH) results from the spontaneous rupture of blood vessels in the brain and represents one of the most devastating and disabling forms of stroke. It accounts for 10–15% of all cases of stroke, showing the highest mortality rate: more than one-third of patients will not survive the first year [ 1 ]. Currently, the lack of effective therapies following ICH prevents better functional outcomes [ 2 ]. ICH induces cerebral angiogenesis around hematoma from 4–7 days post-lesion [ 3 , 4 ], a fact that has been related to motor recovery following ICH [ 5 ]. However, the exact role of endothelium during angiogenesis and neuronal repair following cerebral hemorrhage remains unknown, although endothelial progenitor cells (EPCs) have been suggested as the main players during these processes after stroke [ 6 , 7 ]. EPCs are circulating endothelial cells with the capacity to differentiate into mature endothelial cells and self-renewing [ 7 ]. EPCs are mobilized from their niches to the bloodstream following stroke, reaching the damaged area and carrying out neovascularization and endothelial repair through self-differentiation, paracrine signaling and exosomes [ 8 – 10 ]. Importantly, several clinical studies have reported an improved long-term recovery in stroke patients who had higher numbers of circulating EPCs within the first week after both the ischemic [ 11 – 13 ] and the hemorrhagic [ 14 , 15 ] insult. Therefore, EPCs are a feasible therapeutic target following ICH. The TNF-like weak inducer of apoptosis (TWEAK) is a ligand of the TNF family that can be presented in two forms, as a transmembrane protein and as a soluble ligand (sTWEAK) following furin proteases activity [ 16 ]. TWEAK was initially discovered in cell death-related mechanisms [ 17 ], but subsequent achievements revealed that it controls other activities besides apoptosis, such as proliferation, migration, differentiation, angiogenesis and inflammation [ 18 ]. The binding of sTWEAK to its cellular surface receptor Fn14 triggers several signaling pathways, including the canonical NFκB pathway [ 18 ]. The TWEAK-Fn14 axis regulates several physiological processes, and it is particularly important in tissue repair following acute damage [ 19 ]. Interestingly, Sheng and colleagues [ 20 ] highlighted the relevance of the TWEAK-Fn14-NFκB axis in EPC differentiation, viability, migration to injured tissue and angiogenesis in an in vivo model of acute myocardial infarction. Therefore, TWEAK-mediated mobilization of EPCs could represent a new avenue to promote recovery following ICH. However, the use of TWEAK may be controversial. Several previous studies suggested detrimental effects of endogenous sTWEAK as well as TWEAK treatments in both in vitro and in vivo models of cerebral ischemia [ 21 – 24 ]. There are no preclinical studies addressing the relationship between TWEAK treatment and hemorrhagic stroke so far. The main goal of this proof-of-concept preclinical study was to assess the use of TWEAK as a treatment to provide brain tissue repair through EPCs-mediated neovascularization in a rat model of ICH. MATERIAL AND METHODS Animals All Experimental protocols were approved by the University Clinical Hospital of Santiago de Compostela Animal Care Committee (15010/2019/004), according to the European Union (EU) rules (86/609/CEE, 2003/65/CE and 2010/63/EU) and the ARRIVE guidelines. Male adult Sprague-Dawley (SD) rats (300–350 g) were kept in day/night cycles of 12/12 h at a mean temperature of 22 ± 1°C and humidity of 60 ± 5%, and they had water and food ad libitum . ICH rat model The ICH rat model was used as previously described [ 25 , 26 ]. Anesthesia was maintained by inhalation of 4% sevoflurane in a N 2 O/O 2 mixture (70/30), and body temperature was maintained at 37 ± 0.5°C with a heating pad until animals completely recovered from anesthesia and displayed normal motor activity. Rats were placed in a stereotaxic frame (Stoelting Co, Wood Dale, IL) under sevoflurane anesthesia. After drilling a small burr hole, 1 µL of saline containing 0.2 U/µL bacterial collagenase type VII (Sigma-Aldrich Corp, St. Louis, MO) was injected into the right striatum (0.6 mm anterior to bregma, -3.0 mm lateral and 5.5 mm depth) using a Hamilton syringe with a 30 G needle. Injections took 10 minutes, and the needle was left for an additional 10 minutes before removal. The burr hole was filled with bone wax (Ethicon, Somerville, NJ), and the scalp incision was closed with sutures. Experimental groups Three experimental groups (n = 6 per group) were designated: (1) a control group treated with saline (0.9% of NaCl); (2) 50 group, treated with 50 µg/kg of rat recombinant TWEAK (rTWEAK, #80154-R01H, Sino Biological, Beijing, China) dissolved in saline; and (3) 150 group, treated with 150 µg/kg of rTWEAK dissolved in saline. The concentrations of rTWEAK were selected taking previous work as a reference [ 27 ], but here we wanted to assess potential dose-dependent effects. All treatments were given as a single bolus (jugular) at two timepoints: 1) 1h after ICH, following the basal magnetic resonance imaging (MRI), and 2) 24h after ICH induction. The required sample size was calculated from previous studies using the same model in order to be able to detect a 25% effect size on hematoma growth versus controls (2-tailed t-test) [ 25 , 28 ]. Six animals per group are required to detect this difference with a power (1 − β) of 0.8 and α = 0.05. N was calculated using EPIDAT software ( http://www.sergas.es/Saude-publica/EPIDAT-4-2 ). Animals with hemorrhage located far from the basal ganglia (n = 2) were excluded from the study before treatment administration. The experimental procedure was performed following several criteria derived from the STAIR (Stroke Therapy Academic Industry Roundtable) group guidelines for preclinical evaluation of stroke therapeutics [ 29 ]: (1) ICH hematoma was evaluated at 1 hour, right before rTWEAK injections, by T2-weighted MRI to confirm ICH, as an index of the reliability of the hemorrhagic model; (2) animals were randomly assigned to treatment groups of the study; (3) researchers were blinded to treatment administration; (4) researchers were blinded to treatments during outcome assessment; and (5) temperature was controlled during the surgical period. Magnetic resonance imaging protocol Based on a previous hematoma growth profile study [ 25 , 26 ], hematoma volumes were assessed basally (1 hour after collagenase injection to induce ICH) and at 24 hours, 7, 14, and 28 days after ICH induction by means of MRI conducted on a 9.4-T horizontal bore magnet system (Biospec 94/20USR, Bruker BioSpin, Ettlingen, Germany) with 20-cm-wide actively shielded gradient coils (440 mT/m), as previously described [ 25 , 26 ]. Before MRI acquisition, the animals were placed in a gas chamber containing 6% sevoflurane in a NO 2 /O 2 mixture (70/30) until they were unconscious and then they were positioned prone on the scanner bed. Rectal temperature was maintained at 37 ± 0.5°C using a feedback-controlled heating pad. Radiofrequency transmission was achieved with a birdcage volume resonator, and the signal was detected using a four-element surface coil positioned over the head of the animal. Gradient-echo pilot scans were performed at the beginning of each imaging session for accurate positioning of the animal inside the magnet bore. T2-weighted images were acquired using a Rapid Acquisition Relaxation Enhancement (RARE) sequence with the following acquisition parameters: echo time = 9.5 ms, 8 echos, rare factor = 4, repetition time = 3 seconds, number of averages = 2, field-of view = 19.2 × 19.2 mm 2 , image matrix = 192 × 192 (isotropic in-plane resolution of 0.1 mm 2 /pixel), and 18 consecutive slices of 0.5 mm thickness. All images were processed using ImageJ (RasbandWS, ImageJ, NIH, http://rsb.info.nih.gov/ij ). The analyzed region of interest was the hematoma. Hematoma volumes (basal, 24 hours and 7, 14 and 28 days) as well as edema volumes (24h and 7, 14 and 28 days) were manually traced from T2-weighted images by a blind investigator. Edema was firstly estimated by measuring the volumes of the affected (VLes) and contralateral (Vc) hemispheres and using the formula: edema (%) = 100 × [(VLes − Vc)/Vc]. Then, these values were normalized against those from the 24 h timepoint. Bederson scale Following STAIR criteria, animal models must show neurological and functional deficits in line with the produced lesion. The model of collagenase-induced hemorrhage primarily damages the striatum, producing a small forelimb paresis contralateral to the lesion. Neurological deficit was evaluated using a modified Bederson scale [ 30 ], ranging from 0 (asymptomatic) to 8 (severe deficit), which included the following items: spontaneous movement, spontaneous rotation, spontaneous flexing of the contralateral forelimb, edge detection, turn after tail suspension, protection reflex. Behavioral studies were performed at baseline (before surgery), as well as at 1, 7, and 28 days after ICH during the darkness cycle. An experienced blind investigator analyzed the behavioral tests. Flow cytometry analysis of endothelial progenitor cells Blood samples were drawn from the tail vein before ICH (basal sample), and at days 1, 3, 7, 14, and 28 days after ICH. The samples were collected into K2EDTA tubes (BD Microtainer, USA), then, erythrocytes were lysated using a commercial kit (FACS Lysing, #349202, BD Biosciences, USA). Immunofluorescence cell staining was performed with fluorescent conjugated antibodies anti-ckit (#567471, BD Biosciences, USA) and anti-sca-1 (#CL8934PE, Cederlane, Canada). Cell fluorescence was measured 15 minutes after staining by flow cytometry with BD FACS Aria II (BD, Bioscience, Franklin Lakes, NJ, USA). Numbers of EPCs (ckit + /sca1 + ) were calculated using the FACSDiva software as previously described [ 31 ]. Tissue processing After the completion of the neuroimaging study, three animals per group at 28 days after ICH were euthanized by an overdose of anesthetic (sevoflurane 8%) and perfused with PBS and 4% formaldehyde. Brains were dissected out coronally in three parts and postfixed, in the same fixative solution, overnight at 4°C. Brain blocks were rinsed with 0.1 M phosphate buffer and sequentially immersed in 10%, 20% and 30% (w/v) sucrose in phosphate buffer until they sank. After cryoprotection, 20-µm-thick coronal sections were obtained with a freezing-sliding cryostat (Leica CM 1950 AgProtect; Leica Microsystems, Wetzlar, Germany). Immunofluorescence protocol Sections were rinsed in 0.1 M phosphate buffer (PB) and incubated in 50 mM NH 4 Cl for 30 min. Then, a permeabilization protocol was carried out with 0.3% Triton X-100 (Sigma) in 0.1 M Tris/HCl (pH 8.0) for 10 min. Incubation in the primary antibody solution was carried out in CaCl 2 -containing buffer (0.1 mM CaCl 2 , 0.1 mM MgCl 2 , 0.1 mM MnCl) and blocking solution, 0.05% (v/v) Triton X-100 (Sigma) and 2% (v/v) goat serum (Jackson Immoresearch Laboratories) [ 32 ]. Rabbit anti-Iba1 (#019-19741, 1:200, Wako Chemicals, Neuss, Germany) and anti-IB4 (#L2140, 1:50, Sigma) primary antibodies were used. Sections were incubated for 2 h at room temperature with fluorophore-conjugated secondary antibodies (1:500, Jackson Immunoresearch Laboratories). Nuclei were stained with the commercial monomeric cyanine nucleic acid stain TO-PRO3 (far-red fluorescence; Molecular Probes T3605, Invitrogen) for 10 min. After rinsing with PB, sections were mounted with Fluoromount (Sigma) aqueous mounting medium. Sections were examined with a spectral laser confocal microscope (Leica TSC-SL; Leica Microsystems) with three lasers: multiline Argon (488 nm), Helium-Neon (543 nm) and Helium- Neon (633 nm), and equipped with Å~ 40, Å~ 63 (1.4) HCX PL Apo oil immersion objectives for high-resolution imaging. Immunofluorescence quantifications To quantify the intensity of each immunofluorescence (IF) signal in perilesional cortical regions, the area occupied by IB4 + vessels and Iba1 + were estimated using ImageJ software. All values were normalized against control values. The experimenter was blinded during quantifications. Statistical analyses Data were presented as mean ± S.E.M. Normality of the data was determined by the Shapiro-Wilk normality test. The results of each experiment (lesion volume, EPCs numbers, immunofluorescence and Bederson’s score) were analyzed by a one-way ANOVA (for normally distributed data) or a Kruskal–Wallis test (for non-normally distributed data). Correlation analysis was assessed with the Pearson correlation coefficient test. In Figs. 1 , 2 and 5 , significant values were represented by different numbers of asterisks (vs 150 group) or pounds (vs Control group): * (#) p < 0.05; ** (##) p < 0.01; *** (###) p < 0.001; **** (####) p < 0.0001. Statistical analysis was carried out using Prism 8 (GraphPad software, La Jolla, CA). RESULTS rTWEAK decreases long-term hematoma volume after ICH induction The intraparenchymal injection of collagenase caused an intracerebral hematoma with similar size in all animals at basal time point (Fig. 1 A-A’’, F). The 50 µg/kg dose of rTWEAK showed decreased hematoma at long-term compared with both controls and the 150 group, although differences were statistically significant at 28 days post-injury only vs 150 group (Kruskal-Wallis test, p = 0.040) (Fig. 1 B-E’’, G). In order to investigate the effects of a subacute injection of the treatment (at 24 hours), we also analyzed the reduction of brain damage in relation to this timepoint. Here, the long-term reduction of the 50 µg/kg dose is even clearer compared to both controls (Kruskal-Wallis test, p = 0.132) and the 150 µg/kg dose (Kruskal-Wallis test, p = 0.048) (Fig. 1 B-E’’, H). Edema was reduced in all experimental groups 7 days post-damage (Fig. 1 I); and there was also a reduction in the volume of the ipsilateral hemisphere compared to the contralateral one (negative values) at 14 and 28 days, which was bigger in controls than 50 and 150 groups, but not statistically significant (Fig. 1 I). rTWEAK promotes and maintains long-term EPCs mobilization Our analysis demonstrates that only the 50 µg/kg treatment increased the levels of circulating EPCs at different post-ICH timepoints (Fig. 2 ). Such elevated numbers of EPCs were statistically significant as soon as 72h post-injury (Kruskal-Wallis test, vs Control: p = 0.009), and at 7 (One-way ANOVA test, vs Control: p = 0.013; vs 150: p = 0.045), 14 (One-way ANOVA test, vs Control: p = 0.007; vs 150: p = 0.0004), and 28 days (Kruskal-Wallis test, vs Control: p = 0.004; vs 150: p = 0.010) (Fig. 2 A). Moreover, the peak of circulating EPCs in the 50 group was reached at 7 days post-injury (Fig. 2 A) and this correlates with lower hematoma volumes at this timepoint (Fig. 2 B). rTWEAK enhanced cortical vascularization We performed immunohistochemical analysis targeting the vascular cell marker isolectin-B4 (IB4), which represents a suitable index of vascularization [ 32 , 33 ]. Given that IB4 also labels microglial cells, we used the microglia-specific marker Iba1 to distinguish microglial cells from endothelial cells (Fig. 3 A-E). Regarding IB4 + cells, we observed that only the 50 group had increased vascular density in cortical areas at 28 days post-injury, as revealed by the enhanced IB4 staining indicating vascular repair, and so, neovascularization (One-way ANOVA test, vs Control: p = 0.057; vs 150: p = 0.001) (Fig. 3 B,E and Fig. 4 ). Moreover, the 150 group also displayed statistically significant differences compared to controls (One-way ANOVA test, p = 0.016). The effect of rTWEAK treatments on neurological recovery We used the modified Bederson scale to assess any beneficial effect of rTWEAK treatments on the neurological deficits caused by the hemorrhagic lesion. Results showed that scores were close to 0 at baseline, as expected for healthy subjects (Fig. 5 A); however, both rTWEAK treatments showed higher deficit at 48h (Fig. 5 A), in agreement with the larger hematoma volume seen at 24h. Therefore, we analyzed the effects of a subacute injection (24 hours) and observed that the 50 µg/kg dose, but not 150 µg/kg dose, induced a relevant neurological recovery at post-ICH timepoints compared to both other groups, specially at 28 days (Kruskal-Wallis test, vs 150: p = 0.012) post-injury (Fig. 5 B). DISCUSSION Here we show that a 50 µg/kg dose of rTWEAK induces smaller long-term lesion volumes, mobilizes higher numbers of circulating EPCs, and enhances neovascularization. Thus, our study represents the first proof-of-concept study assessing the therapeutic and dose-effect of TWEAK treatments in an animal model of ICH. Remarkably, our results suggest a direct effect of this treatment on EPCs-mediated vascularization. Here, we discuss the implications and possible mechanisms underlying this TWEAK-mediated response following ICH. The role of the TWEAK-Fn14 axis as an in vitro and in vivo inducer of growth, proliferation, and migration of mature and progenitor endothelial cells is well-known, and it acts in a dose-dependent manner [ 20 , 22 , 34 – 38 ]. Intriguingly, our experiments showed that the rTWEAK treatment of 50 µg/kg mobilizes EPCs to the blood flow in a significant way, whereas the 150 µg/kg concentration had no effect on EPCs, likely because of Fn14 receptor saturation. Furthermore, several previous studies also showed that TWEAK can promote angiogenesis in mature and progenitor endothelial cells both in vitro and in vivo [ 20 , 34 , 35 , 37 ]. Importantly, our experimental group treated with a 50 µg/kg dose of TWEAK showed a higher degree of neovascularization in immunohistochemical analysis at day 28 post-injury, which agrees with previous studies. Based on the important role of EPCs in angiogenesis and neovascularization [ 7 ], it is plausible that a low dose of TWEAK induces EPCs-mediated neovascularization, given that this outcome was not seen in the high dose group, where no significant EPCs mobilization was seen. However, we cannot rule out a potential synergy of TWEAK with other angiogenic factors (e.g. basic fibroblast growth factor [bFGF] and vascular endothelial growth factor-A [VEGF-A]) given that previous studies suggested interactions between them [ 37 , 38 ]. Future studies should confirm whether TWEAK-mediated neovascularization is achieved through EPCs mobilization and/or angiogenic factors. Besides modulating angiogenesis and neovascularization, many studies have addressed the beneficial roles of EPCs following stroke such as reducing inflammation and promoting neuronal survival [ 7 ]. Indeed, higher numbers of circulating EPCs within the first week were associated with an improved long-term recovery in patients who suffered from both ischemic [ 11 – 13 ] and hemorrhagic [ 14 , 15 ] stroke. Here, we observed a higher and sustained EPCs mobilization and neovascularization after the administration of the 50 µg/kg dose, which was reflected in a beneficial long-term outcome both on lesion volumes and on the neurological recovery of animals compared to control counterparts. Furthermore, peak levels of circulating EPCs in the 50 group were reached at 7 days post-injury and were correlated with lower hematoma volumes. Therefore, our results agree with previous studies and highlight the positive role of increasing EPCs following stroke. Results in rodent models of ischemic stroke showed that the blockage of the TWEAK/Fn14 axis results in beneficial effects [ 21 – 24 ]. Similarly, a few clinical studies reported a potential correlation between serum levels of sTWEAK and both a poor functional outcome in ischemic stroke patients [ 39 ] and the risk of developing early ICH growth [ 40 ]. Nevertheless, our strategy was not based on inhibiting TWEAK but on using it in a low dose given that several works indicated that the TWEAK/Fn14 axis can coordinate the inflammation and the response of progenitor cells in the context of acute tissue damage to promote tissue repair [ 41 ]. In this way, present results suggest that our hypothesis was partially right as the 50 µg/kg dose exhibited modest results compared to the control group regarding lesion, edema and behavior but a major impact on EPCs dynamics and angiogenesis. Remarkably, the second injection at 24h post-injury appeared to be crucial in this TWEAK-mediated EPCs mobilization. Indeed, acute injection of rTWEAK at 1h post-injury seems to slightly increase injury volumes at 24h. Such results suggest that the subacute treatment is more efficient than the acute one and so may explain the statistically relevant differences when data is relativized to 24h/48h. Therefore, this indicates that activating the TWEAK/Fn14 axis following hemorrhagic stroke is not harmful per se , but it depends on the intensity and/or the timing. Further studies assessing lower doses than 50 µg/kg of TWEAK at different subacute/chronic timepoints would be necessary to find the best dose and time to apply the treatment. Importantly, the ICH injury may not activate the same molecular pathways as ischemic injury (e.g. the NFκB pathway [ 42 ]), and this could explain the differences between previous ischemic studies and our results. Moreover, the ischemic component in ICH, if so, is clearly less potent than in the ischemic stroke, which implies less apoptosis and tissue destruction [ 26 , 43 ]; further justifying the apparent paradox between previous works and our results. Overall, more studies assessing differences in molecular pathways between ischemic and ICH models are needed. A 150 µg/kg dose of TWEAK increased the hematoma volume compared to control and 50 µg/kg groups at all timepoints after ICH. The intracerebral injection of TWEAK in healthy mice resulted in an increased BBB permeability and accumulation of fluid in the perivascular space, and the inhibition of endogenous TWEAK following ischemic stroke preserved the architecture of the neurovascular unit (NVU) [ 22 ]. These results partially agree with our data despite using different animal models and drug delivery approaches, suggesting that the 150 µg/kg dose exceeded the threshold for beneficial/harmless effects. A previous in vitro study reported the dose-dependent increase in pro-inflammatory cytokines released by astrocytes treated with TWEAK [ 44 ]. Subsequent in vivo studies showed astrocytes as the main targets of endogenous TWEAK that trigger BBB dysfunction after cerebral ischemia [ 22 – 24 ]. Based on our data, we hypothesize that the 150 µg/kg dose is harmful enough to act on and activate NVU-forming astrocytes resulting in bigger lesion volumes without an apparent effect on animal behavior and recovery. Undoubtedly, further studies are mandatory to elucidate the exact impact of TWEAK on astrocytes and BBB dysfunction following ICH. In conclusion, we found that a 50 µg/kg dose of rTWEAK mobilizes higher numbers of circulating EPCs, enhances neovascularization, and induces smaller lesion volumes. Remarkably, our results suggest a direct effect of this treatment on EPCs-mediated vascularization. However, further regulatory preclinical and clinical studies should be conducted to clarify whether rTWEAK may be able to be a therapeutic target in hemorrhagic stroke. Declarations CONFLICTS OF INTEREST The authors declare no conflicts of interest. Author Contribution Conceptualization and design, D.R.-S, E.L.-A. A.O. and T.S.; methodology, D.R.-S, E.L.-A., C.R., P.H., M.R.-A., M.D.-M, R.I.-R.; data analysis, D.R.-S, E.L.-A., C.R., P.H., M.R.-A., M.D.-M, J.M.P.-P., P.A., A.A., J.C., A.O., T.S.; writing the draft, D.R.-S. and T.S., funding acquisition; D.R.-S., A.A., J.C. and T.S. All authors have contributed to editing the manuscript. All authors have revised and approved the final version of the manuscript. Acknowledgments This study was partially supported by grants from the Xunta de Galicia (IN607A2022/07), Instituto de Salud Carlos III (ISCIII) (PI22/00938; PI21/00727; RD21/0006/0005) and CIBERNED (CB22/05/00067). This project has also received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement (No. 101066444). R. Iglesias-Rey (CP22/00061) from the Miguel Servet Program of Instituto de Salud Carlos III and co-financed by the EU. Furthermore, this study was also supported by grants from the INTERREG Atlantic Area (EAPA_791/2018_ NEUROATLANTIC project), INTER-REG V A España Portugal (POCTEP) (0624_2IQBIONEURO_6_E), and the European Regional Development Fund (ERDF). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. References Carhuapoma L, Murthy S, Shah VA (2024) Outcome Trajectories after Intracerebral Hemorrhage. Semin Neurol 44:298–307. 10.1055/s-0044-1787104 Seiffge DJ, Fandler-Höfler S, Du Y et al (2024) Intracerebral haemorrhage - mechanisms, diagnosis and prospects for treatment and prevention. Nat Rev Neurol 20:708–723. 10.1038/s41582-024-01035-w Tang T, Liu XJ, Zhang ZQ et al (2007) Cerebral angiogenesis after collagenase-induced intracerebral hemorrhage in rats. 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J Am Heart Assoc 6:e006042. 10.1161/JAHA.117.006042 Gómez-Lado N, López-Arias E, Iglesias-Rey R et al (2020) [ 18 F]-FMISO PET/MRI Imaging Shows Ischemic Tissue around Hematoma in Intracerebral Hemorrhage. Mol Pharm 17:4667–4675. 10.1021/acs.molpharmaceut.0c00932 Dogra C, Changotra H, Wedhas N, Qin X, Wergedal JE, Kumar A (2007) TNF-related weak inducer of apoptosis (TWEAK) is a potent skeletal muscle-wasting cytokine. FASEB J 21:1857–1869. 10.1096/fj.06-7537com da Silva-Candal A, Vieites-Prado A, Gutiérrez-Fernández M et al (2015) Blood glutamate grabbing does not reduce the hematoma in an intracerebral hemorrhage model but it is a safe excitotoxic treatment modality. J Cereb Blood Flow Metab 35:1206–1212. 10.1038/jcbfm.2015.28 Philip M, Benatar M, Fisher M, Savitz SI (2009) Methodological quality of animal studies of neuroprotective agents currently in phase II/III acute ischemic stroke trials. Stroke 40:577–581. 10.1161/STROKEAHA.108.524330 Bederson JB, Pitts LH, Tsuji M, Nishimura MC, Davis RL, Bartkowski H (1986) Rat middle cerebral artery occlusion: evaluation of the model and development of a neurologic examination. Stroke 17:472–476. 10.1161/01.str.17.3.472 Fadini GP, Sartore S, Schiavon M et al (2006) Diabetes impairs progenitor cell mobilisation after hindlimb ischaemia-reperfusion injury in rats. Diabetologia 49:3075–3084. 10.1007/s00125-006-0401-6 Rodríguez C, Sobrino T, Agulla J et al (2017) Neovascularization and functional recovery after intracerebral hemorrhage is conditioned by the Tp53 Arg72Pro single-nucleotide polymorphism. Cell Death Differ 24:144–154. 10.1038/cdd.2016.109 Wälchli T, Mateos JM, Weinman O et al (2015) Quantitative assessment of angiogenesis, perfused blood vessels and endothelial tip cells in the postnatal mouse brain. Nat Protoc 10:53–74. 10.1038/nprot.2015.002 Lynch CN, Wang YC, Lund JK, Chen YW, Leal JA, Wiley SR (1999) TWEAK induces angiogenesis and proliferation of endothelial cells. J Biol Chem 274:8455–8459. 10.1074/jbc.274.13.8455 Wiley SR, Cassiano L, Lofton T et al (2001) A novel TNF receptor family member binds TWEAK and is implicated in angiogenesis. Immunity 15:837–846. 10.1016/s1074-7613(01)00232-1 Harada N, Nakayama M, Nakano H, Fukuchi Y, Yagita H, Okumura K (2002) Pro-inflammatory effect of TWEAK/Fn14 interaction on human umbilical vein endothelial cells. Biochem Biophys Res Commun 299:488–493. 10.1016/s0006-291x(02)02670-0 Jakubowski A, Browning B, Lukashev M et al (2002) Dual role for TWEAK in angiogenic regulation. J Cell Sci 115:267–274. 10.1242/jcs.115.2.267 Donohue PJ, Richards CM, Brown SA et al (2003) TWEAK is an endothelial cell growth and chemotactic factor that also potentiates FGF-2 and VEGF-A mitogenic activity. Arterioscler Thromb Vasc Biol 23:594–600. 10.1161/01.ATV.0000062883.93715.37 da Silva-Candal A, Pérez-Mato M, Rodríguez-Yáñez M et al (2020) The presence of leukoaraiosis enhances the association between sTWEAK and hemorrhagic transformation. Ann Clin Transl Neurol 7:2103–2114. 10.1002/acn3.51171 da Silva-Candal A, López-Dequidt I, Rodriguez-Yañez M et al (2021) sTWEAK is a marker of early haematoma growth and leukoaraiosis in intracerebral haemorrhage. Stroke Vasc Neurol 6:528–535. 10.1136/svn-2020-000684 Burkly LC, Michaelson JS, Hahm K, Jakubowski A, Zheng TS (2007) TWEAKing tissue remodeling by a multifunctional cytokine: role of TWEAK/Fn14 pathway in health and disease. Cytokine 40:1–16. 10.1016/j.cyto.2007.09.007 Shih RH, Wang CY, Yang CM (2015) NF-kappaB Signaling Pathways in Neurological Inflammation: A Mini Review. Front Mol Neurosci 8:77. 10.3389/fnmol.2015.00077 Xi G, Keep RF, Hoff JT (2006) Mechanisms of brain injury after intracerebral haemorrhage. Lancet Neurol 5:53–63. 10.1016/S1474-4422(05)70283-0 Saas P, Boucraut J, Walker PR et al (2000) TWEAK stimulation of astrocytes and the proinflammatory consequences. Glia 32:102–107 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5796126","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":399927399,"identity":"f5e0a841-e673-4d27-a9c2-34eabb18cae1","order_by":0,"name":"Daniel Romaus-Sanjurjo","email":"data:image/png;base64,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","orcid":"","institution":"Health Research Institute of Santiago de Compostela (IDIS)","correspondingAuthor":true,"prefix":"","firstName":"Daniel","middleName":"","lastName":"Romaus-Sanjurjo","suffix":""},{"id":399927400,"identity":"f450d9d0-f859-411c-8991-520d4d7818cd","order_by":1,"name":"Esteban López-Arias","email":"","orcid":"","institution":"Health Research Institute of Santiago de Compostela (IDIS)","correspondingAuthor":false,"prefix":"","firstName":"Esteban","middleName":"","lastName":"López-Arias","suffix":""},{"id":399927401,"identity":"cbe4f944-644b-4a7a-ad73-04b9b2969a84","order_by":2,"name":"Cristina Rodríguez","email":"","orcid":"","institution":"Instituto de Investigación Biomédica de Salamanca","correspondingAuthor":false,"prefix":"","firstName":"Cristina","middleName":"","lastName":"Rodríguez","suffix":""},{"id":399927402,"identity":"7c428451-5441-45f1-a13d-23e6333a4bce","order_by":3,"name":"Pablo Hervella","email":"","orcid":"","institution":"Health Research Institute of Santiago de Compostela (IDIS)","correspondingAuthor":false,"prefix":"","firstName":"Pablo","middleName":"","lastName":"Hervella","suffix":""},{"id":399927403,"identity":"56bba96a-e4db-4bc5-ab2d-2a46ea402bf6","order_by":4,"name":"Mariña Rodríguez-Arrizabalaga","email":"","orcid":"","institution":"Health Research Institute of Santiago de Compostela (IDIS)","correspondingAuthor":false,"prefix":"","firstName":"Mariña","middleName":"","lastName":"Rodríguez-Arrizabalaga","suffix":""},{"id":399927407,"identity":"516d3828-4649-47a0-96f3-07c0e9d24d47","order_by":5,"name":"Manuel Debasa-Mouce","email":"","orcid":"","institution":"Health Research Institute of Santiago de Compostela (IDIS)","correspondingAuthor":false,"prefix":"","firstName":"Manuel","middleName":"","lastName":"Debasa-Mouce","suffix":""},{"id":399927410,"identity":"c3fe2fd5-dfb0-4d96-b23f-1d2db4b5e1bf","order_by":6,"name":"Juan Manuel Pías-Peleteiro","email":"","orcid":"","institution":"Health Research Institute of Santiago de Compostela (IDIS)","correspondingAuthor":false,"prefix":"","firstName":"Juan","middleName":"Manuel","lastName":"Pías-Peleteiro","suffix":""},{"id":399927414,"identity":"cdbd72ef-73a4-495b-a6ec-dd215b12933e","order_by":7,"name":"Ramón Iglesias-Rey","email":"","orcid":"","institution":"Health Research Institute of Santiago de Compostela (IDIS)","correspondingAuthor":false,"prefix":"","firstName":"Ramón","middleName":"","lastName":"Iglesias-Rey","suffix":""},{"id":399927415,"identity":"9cd69c22-a5b6-466c-90c6-89ede7d35c3f","order_by":8,"name":"Pablo Aguiar","email":"","orcid":"","institution":"University of Santiago de Compostela (USC)","correspondingAuthor":false,"prefix":"","firstName":"Pablo","middleName":"","lastName":"Aguiar","suffix":""},{"id":399927416,"identity":"f8bcd346-96cd-48b0-8ef2-e3c108e0a726","order_by":9,"name":"Ángeles Almeida","email":"","orcid":"","institution":"Instituto de Investigación Biomédica de Salamanca","correspondingAuthor":false,"prefix":"","firstName":"Ángeles","middleName":"","lastName":"Almeida","suffix":""},{"id":399927418,"identity":"ab7ca197-6d5e-4b27-aec7-55eec551efd0","order_by":10,"name":"José Castillo","email":"","orcid":"","institution":"Health Research Institute of Santiago de Compostela (IDIS)","correspondingAuthor":false,"prefix":"","firstName":"José","middleName":"","lastName":"Castillo","suffix":""},{"id":399927419,"identity":"c93bf7dc-f2a9-48c5-8d06-738da7c2ddc6","order_by":11,"name":"Alberto Ouro","email":"","orcid":"","institution":"Health Research Institute of Santiago de Compostela (IDIS)","correspondingAuthor":false,"prefix":"","firstName":"Alberto","middleName":"","lastName":"Ouro","suffix":""},{"id":399927420,"identity":"cbbc4f6a-639b-424f-8fcb-f62cbf4f4e66","order_by":12,"name":"Tomás Sobrino","email":"","orcid":"","institution":"Health Research Institute of Santiago de Compostela (IDIS)","correspondingAuthor":false,"prefix":"","firstName":"Tomás","middleName":"","lastName":"Sobrino","suffix":""}],"badges":[],"createdAt":"2025-01-09 11:38:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5796126/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5796126/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":73461828,"identity":"66247df5-39eb-418e-ab13-13435ba889db","added_by":"auto","created_at":"2025-01-10 08:03:36","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1099794,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTreatment with 50 μg/kg of rTWEAK promotes a reduction of hematoma in the long term.\u003c/strong\u003e (A-E’’) Analysis of hematoma volume was performed by T2-weighted magnetic resonance image. Saline (control) or rTWEAK (50 μg/kg and 150 μg/kg) treatments were administered at 1 and 24 hours after collagenase injection (n=6 per group). (F) Basal hematoma volumes. (G) Percentage of hematoma expansion from basal timepoint. (H) Percentage of hematoma reduction from the 24h timepoint. (I) Analysis of edema evolution from the 24h timepoint. Differences from 150 group vs 50 group are denoted as *p \u0026lt; 0.05. Bars show mean ± SEM.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5796126/v1/43f86d271f9fe808df2341f9.png"},{"id":73461246,"identity":"6fac16c3-d814-42a5-80ff-b959b8af7f18","added_by":"auto","created_at":"2025-01-10 07:55:36","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":204627,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTreatment with 50 μg/kg of rTWEAK promotes EPCs mobilization. \u003c/strong\u003e(A) Time course of endothelial progenitor cells (EPCs: ckit+/sca1+) numbers from blood samples (250.000 cells counted in each one). Stats at each time point are controls (#) and 150 (*) vs. 50 group. *p \u0026lt; 0.05; **p \u0026lt; 0.01; ***p \u0026lt; 0.001. A total of 6 rats per group were used. Bars show mean ± SEM. (B) Negative correlation between number of EPCs and hematoma volume at 7 days post-injury (r = - 0.75; p = 0.086) (n=6).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5796126/v1/9925d9ea0cd40b789d97f7a2.png"},{"id":73461250,"identity":"80b7ce5d-85e9-461a-b9a3-9d4a2a3d5345","added_by":"auto","created_at":"2025-01-10 07:55:36","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1666286,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMicroglia and vascular labeling.\u003c/strong\u003e Saline (control, n=3) or rTWEAK (50 μg/kg and 150 μg/kg, n=3 each group) treated rats were subjected to experimental ICH. Brain sections were co-stained with the vascular endothelial cell marker IB4 and the microglia-specific marker Iba1 at day 28 after experimental ICH. Scale bars: 50 μm. (A-C) Detail from the cortical area (gray box) at higher magnification. Scale bars: 25 μm. Quantification of IB4 (D) and Iba1 (E) immunoreactivity in perilesional cortical regions. Stats: *p = 0.016, Control vs 150 TWEAK; **p = 0.001, 50 TWEAK vs 150 TWEAK. Bars show mean ± SEM.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-5796126/v1/3f68043378b56321aa2dd4ad.png"},{"id":73461829,"identity":"742f5ede-fae7-4844-bde1-18ecc722ad5e","added_by":"auto","created_at":"2025-01-10 08:03:36","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":681115,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNeovascularization after ICH is increased only by the 50 µg/kg rTWEAK treatment.\u003c/strong\u003e Brain sections from the 50 group (n=3) were stained with the vascular endothelial cell marker IB4, the far-red fluorescent nuclear dye TO-PRO3 and the microglia-specific marker Iba1 at day 28 after experimental ICH. Scale bars: 50 μm. Gray box: Detail at higher magnification. Scale bars: 25 μm.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-5796126/v1/83e38f0e3237c9d6b937a0fa.png"},{"id":73461254,"identity":"27bee238-b0aa-4d3a-9810-ce4b9ae87dd6","added_by":"auto","created_at":"2025-01-10 07:55:36","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":157929,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe 50 µg/kg rTWEAK treatments improved long-term neurological recovery after ICH.\u003c/strong\u003e (A) Bederson scores were obtained in control (n=6) and rats treated with the 50 µg/kg dose (n=6) or the 150 µg/kg dose (n=6) at 1 day before surgery (baseline) and 48 hours, 7, and 28 days afterward. (B) Analysis of neurological recovery from 48h timepoint. Differences from 150 group vs 50 group are denoted as *p \u0026lt; 0.05. Bars show mean ± SEM.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-5796126/v1/88ddc65200499bfc513c9f6e.png"},{"id":73709872,"identity":"f9b1ee69-dec7-4c2d-906e-6db707e7c4cc","added_by":"auto","created_at":"2025-01-13 20:31:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4828022,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5796126/v1/9f2edc27-711b-4a69-8417-dffd9946fb4f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eTweak Promotes Neovascularization And Brain Damage Reduction In A Rat Model Of Intracerebral Hemorrhage\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eNon-traumatic intracerebral hemorrhage (ICH) results from the spontaneous rupture of blood vessels in the brain and represents one of the most devastating and disabling forms of stroke. It accounts for 10\u0026ndash;15% of all cases of stroke, showing the highest mortality rate: more than one-third of patients will not survive the first year [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Currently, the lack of effective therapies following ICH prevents better functional outcomes [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eICH induces cerebral angiogenesis around hematoma from 4\u0026ndash;7 days post-lesion [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], a fact that has been related to motor recovery following ICH [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. However, the exact role of endothelium during angiogenesis and neuronal repair following cerebral hemorrhage remains unknown, although endothelial progenitor cells (EPCs) have been suggested as the main players during these processes after stroke [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. EPCs are circulating endothelial cells with the capacity to differentiate into mature endothelial cells and self-renewing [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. EPCs are mobilized from their niches to the bloodstream following stroke, reaching the damaged area and carrying out neovascularization and endothelial repair through self-differentiation, paracrine signaling and exosomes [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Importantly, several clinical studies have reported an improved long-term recovery in stroke patients who had higher numbers of circulating EPCs within the first week after both the ischemic [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] and the hemorrhagic [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] insult. Therefore, EPCs are a feasible therapeutic target following ICH.\u003c/p\u003e \u003cp\u003eThe TNF-like weak inducer of apoptosis (TWEAK) is a ligand of the TNF family that can be presented in two forms, as a transmembrane protein and as a soluble ligand (sTWEAK) following furin proteases activity [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. TWEAK was initially discovered in cell death-related mechanisms [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], but subsequent achievements revealed that it controls other activities besides apoptosis, such as proliferation, migration, differentiation, angiogenesis and inflammation [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The binding of sTWEAK to its cellular surface receptor Fn14 triggers several signaling pathways, including the canonical NFκB pathway [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The TWEAK-Fn14 axis regulates several physiological processes, and it is particularly important in tissue repair following acute damage [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Interestingly, Sheng and colleagues [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] highlighted the relevance of the TWEAK-Fn14-NFκB axis in EPC differentiation, viability, migration to injured tissue and angiogenesis in an \u003cem\u003ein vivo\u003c/em\u003e model of acute myocardial infarction. Therefore, TWEAK-mediated mobilization of EPCs could represent a new avenue to promote recovery following ICH. However, the use of TWEAK may be controversial. Several previous studies suggested detrimental effects of endogenous sTWEAK as well as TWEAK treatments in both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e models of cerebral ischemia [\u003cspan additionalcitationids=\"CR22 CR23\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. There are no preclinical studies addressing the relationship between TWEAK treatment and hemorrhagic stroke so far.\u003c/p\u003e \u003cp\u003eThe main goal of this proof-of-concept preclinical study was to assess the use of TWEAK as a treatment to provide brain tissue repair through EPCs-mediated neovascularization in a rat model of ICH.\u003c/p\u003e"},{"header":"MATERIAL AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003e All Experimental protocols were approved by the University Clinical Hospital of Santiago de Compostela Animal Care Committee (15010/2019/004), according to the European Union (EU) rules (86/609/CEE, 2003/65/CE and 2010/63/EU) and the ARRIVE guidelines. Male adult Sprague-Dawley (SD) rats (300\u0026ndash;350 g) were kept in day/night cycles of 12/12 h at a mean temperature of 22\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C and humidity of 60\u0026thinsp;\u0026plusmn;\u0026thinsp;5%, and they had water and food \u003cem\u003ead libitum\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eICH rat model\u003c/h3\u003e\n\u003cp\u003eThe ICH rat model was used as previously described [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Anesthesia was maintained by inhalation of 4% sevoflurane in a N\u003csub\u003e2\u003c/sub\u003eO/O\u003csub\u003e2\u003c/sub\u003e mixture (70/30), and body temperature was maintained at 37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u0026deg;C with a heating pad until animals completely recovered from anesthesia and displayed normal motor activity. Rats were placed in a stereotaxic frame (Stoelting Co, Wood Dale, IL) under sevoflurane anesthesia. After drilling a small burr hole, 1 \u0026micro;L of saline containing 0.2 U/\u0026micro;L bacterial collagenase type VII (Sigma-Aldrich Corp, St. Louis, MO) was injected into the right striatum (0.6 mm anterior to bregma, -3.0 mm lateral and 5.5 mm depth) using a Hamilton syringe with a 30 G needle. Injections took 10 minutes, and the needle was left for an additional 10 minutes before removal. The burr hole was filled with bone wax (Ethicon, Somerville, NJ), and the scalp incision was closed with sutures.\u003c/p\u003e\n\u003ch3\u003eExperimental groups\u003c/h3\u003e\n\u003cp\u003eThree experimental groups (n\u0026thinsp;=\u0026thinsp;6 per group) were designated: (1) a control group treated with saline (0.9% of NaCl); (2) 50 group, treated with 50 \u0026micro;g/kg of rat recombinant TWEAK (rTWEAK, #80154-R01H, Sino Biological, Beijing, China) dissolved in saline; and (3) 150 group, treated with 150 \u0026micro;g/kg of rTWEAK dissolved in saline. The concentrations of rTWEAK were selected taking previous work as a reference [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], but here we wanted to assess potential dose-dependent effects. All treatments were given as a single bolus (jugular) at two timepoints: 1) 1h after ICH, following the basal magnetic resonance imaging (MRI), and 2) 24h after ICH induction. The required sample size was calculated from previous studies using the same model in order to be able to detect a 25% effect size on hematoma growth versus controls (2-tailed t-test) [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Six animals per group are required to detect this difference with a power (1\u0026thinsp;\u0026minus;\u0026thinsp;β) of 0.8 and α\u0026thinsp;=\u0026thinsp;0.05. N was calculated using EPIDAT software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.sergas.es/Saude-publica/EPIDAT-4-2\u003c/span\u003e\u003cspan address=\"http://www.sergas.es/Saude-publica/EPIDAT-4-2\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Animals with hemorrhage located far from the basal ganglia (n\u0026thinsp;=\u0026thinsp;2) were excluded from the study before treatment administration.\u003c/p\u003e \u003cp\u003eThe experimental procedure was performed following several criteria derived from the STAIR (Stroke Therapy Academic Industry Roundtable) group guidelines for preclinical evaluation of stroke therapeutics [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]: (1) ICH hematoma was evaluated at 1 hour, right before rTWEAK injections, by T2-weighted MRI to confirm ICH, as an index of the reliability of the hemorrhagic model; (2) animals were randomly assigned to treatment groups of the study; (3) researchers were blinded to treatment administration; (4) researchers were blinded to treatments during outcome assessment; and (5) temperature was controlled during the surgical period.\u003c/p\u003e\n\u003ch3\u003eMagnetic resonance imaging protocol\u003c/h3\u003e\n\u003cp\u003eBased on a previous hematoma growth profile study [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], hematoma volumes were assessed basally (1 hour after collagenase injection to induce ICH) and at 24 hours, 7, 14, and 28 days after ICH induction by means of MRI conducted on a 9.4-T horizontal bore magnet system (Biospec 94/20USR, Bruker BioSpin, Ettlingen, Germany) with 20-cm-wide actively shielded gradient coils (440 mT/m), as previously described [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Before MRI acquisition, the animals were placed in a gas chamber containing 6% sevoflurane in a NO\u003csub\u003e2\u003c/sub\u003e/O\u003csub\u003e2\u003c/sub\u003e mixture (70/30) until they were unconscious and then they were positioned prone on the scanner bed. Rectal temperature was maintained at 37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u0026deg;C using a feedback-controlled heating pad. Radiofrequency transmission was achieved with a birdcage volume resonator, and the signal was detected using a four-element surface coil positioned over the head of the animal. Gradient-echo pilot scans were performed at the beginning of each imaging session for accurate positioning of the animal inside the magnet bore.\u003c/p\u003e \u003cp\u003eT2-weighted images were acquired using a Rapid Acquisition Relaxation Enhancement (RARE) sequence with the following acquisition parameters: echo time\u0026thinsp;=\u0026thinsp;9.5 ms, 8 echos, rare factor\u0026thinsp;=\u0026thinsp;4, repetition time\u0026thinsp;=\u0026thinsp;3 seconds, number of averages\u0026thinsp;=\u0026thinsp;2, field-of view\u0026thinsp;=\u0026thinsp;19.2 \u0026times; 19.2 mm\u003csup\u003e2\u003c/sup\u003e, image matrix\u0026thinsp;=\u0026thinsp;192 \u0026times; 192 (isotropic in-plane resolution of 0.1 mm\u003csup\u003e2\u003c/sup\u003e/pixel), and 18 consecutive slices of 0.5 mm thickness. All images were processed using ImageJ (RasbandWS, ImageJ, NIH, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://rsb.info.nih.gov/ij\u003c/span\u003e\u003cspan address=\"http://rsb.info.nih.gov/ij\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The analyzed region of interest was the hematoma. Hematoma volumes (basal, 24 hours and 7, 14 and 28 days) as well as edema volumes (24h and 7, 14 and 28 days) were manually traced from T2-weighted images by a blind investigator.\u003c/p\u003e \u003cp\u003eEdema was firstly estimated by measuring the volumes of the affected (VLes) and contralateral (Vc) hemispheres and using the formula: edema (%)\u0026thinsp;=\u0026thinsp;100 \u0026times; [(VLes\u0026thinsp;\u0026minus;\u0026thinsp;Vc)/Vc]. Then, these values were normalized against those from the 24 h timepoint.\u003c/p\u003e\n\u003ch3\u003eBederson scale\u003c/h3\u003e\n\u003cp\u003eFollowing STAIR criteria, animal models must show neurological and functional deficits in line with the produced lesion. The model of collagenase-induced hemorrhage primarily damages the striatum, producing a small forelimb paresis contralateral to the lesion. Neurological deficit was evaluated using a modified Bederson scale [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], ranging from 0 (asymptomatic) to 8 (severe deficit), which included the following items: spontaneous movement, spontaneous rotation, spontaneous flexing of the contralateral forelimb, edge detection, turn after tail suspension, protection reflex.\u003c/p\u003e \u003cp\u003eBehavioral studies were performed at baseline (before surgery), as well as at 1, 7, and 28 days after ICH during the darkness cycle. An experienced blind investigator analyzed the behavioral tests.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry analysis of endothelial progenitor cells\u003c/h2\u003e \u003cp\u003eBlood samples were drawn from the tail vein before ICH (basal sample), and at days 1, 3, 7, 14, and 28 days after ICH. The samples were collected into K2EDTA tubes (BD Microtainer, USA), then, erythrocytes were lysated using a commercial kit (FACS Lysing, #349202, BD Biosciences, USA). Immunofluorescence cell staining was performed with fluorescent conjugated antibodies anti-ckit (#567471, BD Biosciences, USA) and anti-sca-1 (#CL8934PE, Cederlane, Canada). Cell fluorescence was measured 15 minutes after staining by flow cytometry with BD FACS Aria II (BD, Bioscience, Franklin Lakes, NJ, USA). Numbers of EPCs (ckit\u003csup\u003e+\u003c/sup\u003e/sca1\u003csup\u003e+\u003c/sup\u003e) were calculated using the FACSDiva software as previously described [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTissue processing\u003c/h3\u003e\n\u003cp\u003eAfter the completion of the neuroimaging study, three animals per group at 28 days after ICH were euthanized by an overdose of anesthetic (sevoflurane 8%) and perfused with PBS and 4% formaldehyde. Brains were dissected out coronally in three parts and postfixed, in the same fixative solution, overnight at 4\u0026deg;C. Brain blocks were rinsed with 0.1 M phosphate buffer and sequentially immersed in 10%, 20% and 30% (w/v) sucrose in phosphate buffer until they sank. After cryoprotection, 20-\u0026micro;m-thick coronal sections were obtained with a freezing-sliding cryostat (Leica CM 1950 AgProtect; Leica Microsystems, Wetzlar, Germany).\u003c/p\u003e\n\u003ch3\u003eImmunofluorescence protocol\u003c/h3\u003e\n\u003cp\u003eSections were rinsed in 0.1 M phosphate buffer (PB) and incubated in 50 mM NH\u003csub\u003e4\u003c/sub\u003eCl for 30 min. Then, a permeabilization protocol was carried out with 0.3% Triton X-100 (Sigma) in 0.1 M Tris/HCl (pH 8.0) for 10 min. Incubation in the primary antibody solution was carried out in CaCl\u003csub\u003e2\u003c/sub\u003e-containing buffer (0.1 mM CaCl\u003csub\u003e2\u003c/sub\u003e, 0.1 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 0.1 mM MnCl) and blocking solution, 0.05% (v/v) Triton X-100 (Sigma) and 2% (v/v) goat serum (Jackson Immoresearch Laboratories) [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Rabbit anti-Iba1 (#019-19741, 1:200, Wako Chemicals, Neuss, Germany) and anti-IB4 (#L2140, 1:50, Sigma) primary antibodies were used. Sections were incubated for 2 h at room temperature with fluorophore-conjugated secondary antibodies (1:500, Jackson Immunoresearch Laboratories). Nuclei were stained with the commercial monomeric cyanine nucleic acid stain TO-PRO3 (far-red fluorescence; Molecular Probes T3605, Invitrogen) for 10 min. After rinsing with PB, sections were mounted with Fluoromount (Sigma) aqueous mounting medium. Sections were examined with a spectral laser confocal microscope (Leica TSC-SL; Leica Microsystems) with three lasers: multiline Argon (488 nm), Helium-Neon (543 nm) and Helium- Neon (633 nm), and equipped with \u0026Aring;~ 40, \u0026Aring;~ 63 (1.4) HCX PL Apo oil immersion objectives for high-resolution imaging.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence quantifications\u003c/h2\u003e \u003cp\u003eTo quantify the intensity of each immunofluorescence (IF) signal in perilesional cortical regions, the area occupied by IB4\u003csup\u003e+\u003c/sup\u003e vessels and Iba1\u003csup\u003e+\u003c/sup\u003e were estimated using ImageJ software. All values were normalized against control values. The experimenter was blinded during quantifications.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analyses\u003c/h2\u003e \u003cp\u003eData were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;S.E.M. Normality of the data was determined by the Shapiro-Wilk normality test. The results of each experiment (lesion volume, EPCs numbers, immunofluorescence and Bederson\u0026rsquo;s score) were analyzed by a one-way ANOVA (for normally distributed data) or a Kruskal\u0026ndash;Wallis test (for non-normally distributed data). Correlation analysis was assessed with the Pearson correlation coefficient test. In Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003e, significant values were represented by different numbers of asterisks (vs 150 group) or pounds (vs Control group): *\u003csup\u003e(#)\u003c/sup\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05; **\u003csup\u003e(##)\u003c/sup\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.01; ***\u003csup\u003e(###)\u003c/sup\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001; ****\u003csup\u003e(####)\u003c/sup\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.0001. Statistical analysis was carried out using Prism 8 (GraphPad software, La Jolla, CA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003erTWEAK decreases long-term hematoma volume after ICH induction\u003c/h2\u003e \u003cp\u003eThe intraparenchymal injection of collagenase caused an intracerebral hematoma with similar size in all animals at basal time point (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-A\u0026rsquo;\u0026rsquo;, F). The 50 \u0026micro;g/kg dose of rTWEAK showed decreased hematoma at long-term compared with both controls and the 150 group, although differences were statistically significant at 28 days post-injury only vs 150 group (Kruskal-Wallis test, p\u0026thinsp;=\u0026thinsp;0.040) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-E\u0026rsquo;\u0026rsquo;, G). In order to investigate the effects of a subacute injection of the treatment (at 24 hours), we also analyzed the reduction of brain damage in relation to this timepoint. Here, the long-term reduction of the 50 \u0026micro;g/kg dose is even clearer compared to both controls (Kruskal-Wallis test, p\u0026thinsp;=\u0026thinsp;0.132) and the 150 \u0026micro;g/kg dose (Kruskal-Wallis test, p\u0026thinsp;=\u0026thinsp;0.048) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-E\u0026rsquo;\u0026rsquo;, H). Edema was reduced in all experimental groups 7 days post-damage (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI); and there was also a reduction in the volume of the ipsilateral hemisphere compared to the contralateral one (negative values) at 14 and 28 days, which was bigger in controls than 50 and 150 groups, but not statistically significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003erTWEAK promotes and maintains long-term EPCs mobilization\u003c/h2\u003e \u003cp\u003eOur analysis demonstrates that only the 50 \u0026micro;g/kg treatment increased the levels of circulating EPCs at different post-ICH timepoints (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Such elevated numbers of EPCs were statistically significant as soon as 72h post-injury (Kruskal-Wallis test, vs Control: p\u0026thinsp;=\u0026thinsp;0.009), and at 7 (One-way ANOVA test, vs Control: p\u0026thinsp;=\u0026thinsp;0.013; vs 150: p\u0026thinsp;=\u0026thinsp;0.045), 14 (One-way ANOVA test, vs Control: p\u0026thinsp;=\u0026thinsp;0.007; vs 150: p\u0026thinsp;=\u0026thinsp;0.0004), and 28 days (Kruskal-Wallis test, vs Control: p\u0026thinsp;=\u0026thinsp;0.004; vs 150: p\u0026thinsp;=\u0026thinsp;0.010) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Moreover, the peak of circulating EPCs in the 50 group was reached at 7 days post-injury (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) and this correlates with lower hematoma volumes at this timepoint (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003erTWEAK enhanced cortical vascularization\u003c/h2\u003e \u003cp\u003eWe performed immunohistochemical analysis targeting the vascular cell marker isolectin-B4 (IB4), which represents a suitable index of vascularization [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Given that IB4 also labels microglial cells, we used the microglia-specific marker Iba1 to distinguish microglial cells from endothelial cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-E). Regarding IB4\u003csup\u003e+\u003c/sup\u003e cells, we observed that only the 50 group had increased vascular density in cortical areas at 28 days post-injury, as revealed by the enhanced IB4 staining indicating vascular repair, and so, neovascularization (One-way ANOVA test, vs Control: p\u0026thinsp;=\u0026thinsp;0.057; vs 150: p\u0026thinsp;=\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eB,E and Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Moreover, the 150 group also displayed statistically significant differences compared to controls (One-way ANOVA test, p\u0026thinsp;=\u0026thinsp;0.016).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eThe effect of rTWEAK treatments on neurological recovery\u003c/h2\u003e \u003cp\u003eWe used the modified Bederson scale to assess any beneficial effect of rTWEAK treatments on the neurological deficits caused by the hemorrhagic lesion. Results showed that scores were close to 0 at baseline, as expected for healthy subjects (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eA); however, both rTWEAK treatments showed higher deficit at 48h (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), in agreement with the larger hematoma volume seen at 24h. Therefore, we analyzed the effects of a subacute injection (24 hours) and observed that the 50 \u0026micro;g/kg dose, but not 150 \u0026micro;g/kg dose, induced a relevant neurological recovery at post-ICH timepoints compared to both other groups, specially at 28 days (Kruskal-Wallis test, vs 150: p\u0026thinsp;=\u0026thinsp;0.012) post-injury (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eHere we show that a 50 \u0026micro;g/kg dose of rTWEAK induces smaller long-term lesion volumes, mobilizes higher numbers of circulating EPCs, and enhances neovascularization. Thus, our study represents the first proof-of-concept study assessing the therapeutic and dose-effect of TWEAK treatments in an animal model of ICH. Remarkably, our results suggest a direct effect of this treatment on EPCs-mediated vascularization. Here, we discuss the implications and possible mechanisms underlying this TWEAK-mediated response following ICH.\u003c/p\u003e \u003cp\u003eThe role of the TWEAK-Fn14 axis as an \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e inducer of growth, proliferation, and migration of mature and progenitor endothelial cells is well-known, and it acts in a dose-dependent manner [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan additionalcitationids=\"CR35 CR36 CR37\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Intriguingly, our experiments showed that the rTWEAK treatment of 50 \u0026micro;g/kg mobilizes EPCs to the blood flow in a significant way, whereas the 150 \u0026micro;g/kg concentration had no effect on EPCs, likely because of Fn14 receptor saturation. Furthermore, several previous studies also showed that TWEAK can promote angiogenesis in mature and progenitor endothelial cells both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Importantly, our experimental group treated with a 50 \u0026micro;g/kg dose of TWEAK showed a higher degree of neovascularization in immunohistochemical analysis at day 28 post-injury, which agrees with previous studies. Based on the important role of EPCs in angiogenesis and neovascularization [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], it is plausible that a low dose of TWEAK induces EPCs-mediated neovascularization, given that this outcome was not seen in the high dose group, where no significant EPCs mobilization was seen. However, we cannot rule out a potential synergy of TWEAK with other angiogenic factors (e.g. basic fibroblast growth factor [bFGF] and vascular endothelial growth factor-A [VEGF-A]) given that previous studies suggested interactions between them [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Future studies should confirm whether TWEAK-mediated neovascularization is achieved through EPCs mobilization and/or angiogenic factors.\u003c/p\u003e \u003cp\u003eBesides modulating angiogenesis and neovascularization, many studies have addressed the beneficial roles of EPCs following stroke such as reducing inflammation and promoting neuronal survival [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Indeed, higher numbers of circulating EPCs within the first week were associated with an improved long-term recovery in patients who suffered from both ischemic [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] and hemorrhagic [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] stroke. Here, we observed a higher and sustained EPCs mobilization and neovascularization after the administration of the 50 \u0026micro;g/kg dose, which was reflected in a beneficial long-term outcome both on lesion volumes and on the neurological recovery of animals compared to control counterparts. Furthermore, peak levels of circulating EPCs in the 50 group were reached at 7 days post-injury and were correlated with lower hematoma volumes. Therefore, our results agree with previous studies and highlight the positive role of increasing EPCs following stroke.\u003c/p\u003e \u003cp\u003eResults in rodent models of ischemic stroke showed that the blockage of the TWEAK/Fn14 axis results in beneficial effects [\u003cspan additionalcitationids=\"CR22 CR23\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Similarly, a few clinical studies reported a potential correlation between serum levels of sTWEAK and both a poor functional outcome in ischemic stroke patients [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] and the risk of developing early ICH growth [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Nevertheless, our strategy was not based on inhibiting TWEAK but on using it in a low dose given that several works indicated that the TWEAK/Fn14 axis can coordinate the inflammation and the response of progenitor cells in the context of acute tissue damage to promote tissue repair [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. In this way, present results suggest that our hypothesis was partially right as the 50 \u0026micro;g/kg dose exhibited modest results compared to the control group regarding lesion, edema and behavior but a major impact on EPCs dynamics and angiogenesis. Remarkably, the second injection at 24h post-injury appeared to be crucial in this TWEAK-mediated EPCs mobilization. Indeed, acute injection of rTWEAK at 1h post-injury seems to slightly increase injury volumes at 24h. Such results suggest that the subacute treatment is more efficient than the acute one and so may explain the statistically relevant differences when data is relativized to 24h/48h. Therefore, this indicates that activating the TWEAK/Fn14 axis following hemorrhagic stroke is not harmful \u003cem\u003eper se\u003c/em\u003e, but it depends on the intensity and/or the timing. Further studies assessing lower doses than 50 \u0026micro;g/kg of TWEAK at different subacute/chronic timepoints would be necessary to find the best dose and time to apply the treatment.\u003c/p\u003e \u003cp\u003eImportantly, the ICH injury may not activate the same molecular pathways as ischemic injury (e.g. the NFκB pathway [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]), and this could explain the differences between previous ischemic studies and our results. Moreover, the ischemic component in ICH, if so, is clearly less potent than in the ischemic stroke, which implies less apoptosis and tissue destruction [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]; further justifying the apparent paradox between previous works and our results. Overall, more studies assessing differences in molecular pathways between ischemic and ICH models are needed.\u003c/p\u003e \u003cp\u003eA 150 \u0026micro;g/kg dose of TWEAK increased the hematoma volume compared to control and 50 \u0026micro;g/kg groups at all timepoints after ICH. The intracerebral injection of TWEAK in healthy mice resulted in an increased BBB permeability and accumulation of fluid in the perivascular space, and the inhibition of endogenous TWEAK following ischemic stroke preserved the architecture of the neurovascular unit (NVU) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. These results partially agree with our data despite using different animal models and drug delivery approaches, suggesting that the 150 \u0026micro;g/kg dose exceeded the threshold for beneficial/harmless effects. A previous \u003cem\u003ein vitro\u003c/em\u003e study reported the dose-dependent increase in pro-inflammatory cytokines released by astrocytes treated with TWEAK [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Subsequent \u003cem\u003ein vivo\u003c/em\u003e studies showed astrocytes as the main targets of endogenous TWEAK that trigger BBB dysfunction after cerebral ischemia [\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Based on our data, we hypothesize that the 150 \u0026micro;g/kg dose is harmful enough to act on and activate NVU-forming astrocytes resulting in bigger lesion volumes without an apparent effect on animal behavior and recovery. Undoubtedly, further studies are mandatory to elucidate the exact impact of TWEAK on astrocytes and BBB dysfunction following ICH.\u003c/p\u003e \u003cp\u003eIn conclusion, we found that a 50 \u0026micro;g/kg dose of rTWEAK mobilizes higher numbers of circulating EPCs, enhances neovascularization, and induces smaller lesion volumes. Remarkably, our results suggest a direct effect of this treatment on EPCs-mediated vascularization. However, further regulatory preclinical and clinical studies should be conducted to clarify whether rTWEAK may be able to be a therapeutic target in hemorrhagic stroke.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCONFLICTS OF INTEREST\u003c/h2\u003e \u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization and design, D.R.-S, E.L.-A. A.O. and T.S.; methodology, D.R.-S, E.L.-A., C.R., P.H., M.R.-A., M.D.-M, R.I.-R.; data analysis, D.R.-S, E.L.-A., C.R., P.H., M.R.-A., M.D.-M, J.M.P.-P., P.A., A.A., J.C., A.O., T.S.; writing the draft, D.R.-S. and T.S., funding acquisition; D.R.-S., A.A., J.C. and T.S. All authors have contributed to editing the manuscript. All authors have revised and approved the final version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eThis study was partially supported by grants from the Xunta de Galicia (IN607A2022/07), Instituto de Salud Carlos III (ISCIII) (PI22/00938; PI21/00727; RD21/0006/0005) and CIBERNED (CB22/05/00067). This project has also received funding from the European Union\u0026rsquo;s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement (No. 101066444). R. Iglesias-Rey (CP22/00061) from the Miguel Servet Program of Instituto de Salud Carlos III and co-financed by the EU. Furthermore, this study was also supported by grants from the INTERREG Atlantic Area (EAPA_791/2018_ NEUROATLANTIC project), INTER-REG V A Espa\u0026ntilde;a Portugal (POCTEP) (0624_2IQBIONEURO_6_E), and the European Regional Development Fund (ERDF). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCarhuapoma L, Murthy S, Shah VA (2024) Outcome Trajectories after Intracerebral Hemorrhage. 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Glia 32:102\u0026ndash;107\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"endothelial progenitor cells, intracerebral hemorrhage, neovascularization, TWEAK","lastPublishedDoi":"10.21203/rs.3.rs-5796126/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5796126/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNon-traumatic intracerebral hemorrhage (ICH) is one of the most devastating and disabling forms of stroke; however, there are no effective pharmacological therapies following the insult. Angiogenesis appears as a key step to overcome the damage and promote functional recovery. In this context, endothelial progenitor cells (EPCs) mobilization promotes neovascularization which has been linked to beneficial outcomes following both ischemic and hemorrhagic stroke. The TNF-like weak inducer of apoptosis (TWEAK), binding to its receptor Fn14, has been suggested as an inducer of EPCs differentiation, viability and migration to the injury site in a model of myocardial infarction. Here, we have performed a proof-of-concept preclinical study in a rat model of ICH where we report that a 50 \u0026micro;g/kg dose of rat recombinant TWEAK (rTWEAK) promotes EPCs mobilization, as soon as 72 h post-injury, brain neovascularization, and, importantly, long-term hematoma reduction and functional recovery. In contrast, a higher dose of 150 \u0026micro;g/kg blocked those beneficial outcomes. Therefore, a low-dose of rTWEAK treatment promotes neovascularization and reduces brain damage in a rat model of ICH. Further clinical studies will be needed to demonstrate if rTWEAK could represent a new strategy to promote recovery following ICH.\u003c/p\u003e","manuscriptTitle":"Tweak Promotes Neovascularization And Brain Damage Reduction In A Rat Model Of Intracerebral Hemorrhage","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-10 07:55:31","doi":"10.21203/rs.3.rs-5796126/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f05f393e-a9bb-4bac-b480-f36730aaa654","owner":[],"postedDate":"January 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-01-13T20:23:21+00:00","versionOfRecord":[],"versionCreatedAt":"2025-01-10 07:55:31","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5796126","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5796126","identity":"rs-5796126","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}