antioxidants Article Targeting Pro-Oxidant Iron with Exogenously Administered Apotransferrin Provides Benefits Associated with Changes in Crucial Cellular Iron Gate Protein TfR in a Model of Intracerebral Hemorrhagic Stroke in Mice Alexia García-Serran 1 , Jesús Ordoño 1 , Núria DeGregorio-Rocasolano 1 , Marc Melià-Sorolla 1 , Karla Odendaal 1,2 , Octavi Martí-Sistac 1,3, * and Teresa Gasull 1, * 1 Cellular and Molecular Neurobiology Research Group, Fundació Institut d’Investigació en Ciències de la Salut Germans Trias i Pujol (IGTP), Universitat Autònoma de Barcelona (UAB), 08916 Badalona, Catalonia, Spain; agarcias@igtp.cat (A.G.-S.); jesus.ordono@imdea.org (J.O.); ndgregorio@igtp.cat (N.D.-R.); mmelia@igtp.cat (M.M.-S.); kk.o@btinternet.com (K.O.) 2 School of Biosciences, University of Cardiff, Cardiff CF10 3AT, UK 3 Department of Cellular Biology, Physiology and Immunology, Universitat Autònoma de Barcelona, 08193 Bellaterra, Catalonia, Spain * Correspondence: octavi.marti@uab.cat or omarti@igtp.cat (O.M.-S.); tgasull@igtp.cat or teresagasull@yahoo.com (T.G.); Tel.: +34-93-0330-531 (T.G.) Abstract: We have previously demonstrated that the post-stroke administration of iron-free transfer- rin (apotransferrin, ATf) is beneficial in different models of ischemic stroke (IS) through the inhibition of the neuronal uptake of pro-oxidant iron. In the present study, we asked whether ATf is safe and also beneficial when given after the induction of intracerebral hemorrhage (ICH) in mice, and Citation: García-Serran, A.; Ordoño, J.; DeGregorio-Rocasolano, N.; investigated the underlying mechanisms. We first compared the main iron actors in the brain of IS- or Melià-Sorolla, M.; Odendaal, K.; collagenase-induced ICH mice and then obtained insight into these iron-related proteins in ICH 72 h Martí-Sistac, O.; Gasull, T. Targeting after the administration of ATf. The infarct size of the IS mice was double that of hemorrhage in ICH Pro-Oxidant Iron with Exogenously mice, but both groups showed similar body weight loss, edema, and increased ferritin and transferrin Administered Apotransferrin levels in the ipsilateral brain hemisphere. Although the administration of human ATf (hATf) to ICH Provides Benefits Associated with mice did not alter the hemorrhage volume or levels of the classical ferroptosis GPX4/system xc- Changes in Crucial Cellular Iron Gate pathways, hATf induced better neurobehavioral performance, decreased 4-hydroxynonenal levels Protein TfR in a Model of and those of the second-generation ferroptosis marker transferrin receptor (TfR), and restored the Intracerebral Hemorrhagic Stroke in mRNA levels of the recently recognized cytosolic iron chaperone poly(RC) binding protein 2. In Mice. Antioxidants 2023, 12, 1945. addition, hATf treatment lowered the ICH-induced increase in both endogenous mouse transferrin https://doi.org/10.3390/ mRNA levels and the activation of caspase-2. In conclusion, hATf treatment provides neurobehavioral antiox12111945 benefits post-ICH associated with the modulation of iron/oxidative players. Academic Editors: Syed Shadab Raza and Johannes Boltze Keywords: ischemic stroke; intracerebral hemorrhage; iron; oxidation; apotransferrin; mouse Received: 13 August 2023 Revised: 29 September 2023 Accepted: 9 October 2023 Published: 31 October 2023 1. Introduction A global consensus exists throughout the literature reporting the detrimental effects of systemic iron overload in ischemic stroke, and the protective effect of iron chelators and the iron-binding protein apotransferrin (ATf) in ischemic stroke models [1–3] and ischemic Copyright: © 2023 by the authors. stroke patients [4]. Moreover, iron chelators and ATf have been studied as neuroprotectors Licensee MDPI, Basel, Switzerland. This article is an open access article in hemorrhagic stroke models. Chen-Roetling J et al. reported the protective effect of ATf distributed under the terms and in cortical neurons [5], and deferoxamine has been reported to be beneficial in hemorrhagic conditions of the Creative Commons stroke patients [6,7]. In preclinical studies, our laboratory has shown that ATf, the iron-free Attribution (CC BY) license (https:// form of transferrin (Tf), is a potent neuroprotective agent in ischemic stroke because it creativecommons.org/licenses/by/ reduces the saturation of blood transferrin with iron (TSAT) and inhibits the entry of iron-Tf 4.0/). into the neurons [3]. Antioxidants 2023, 12, 1945. https://doi.org/10.3390/antiox12111945 https://www.mdpi.com/journal/antioxidants Antioxidants 2023, 12, 1945 2 of 18 Intracerebral hemorrhage (ICH), besides the damage it induces through an increase in intracranial pressure derived from a sudden accumulation of spilled blood in the brain parenchyma, brings about toxic effects produced by substances of the leaked blood [8,9]. In particular, the breakdown of erythrocytes leads to the release of hemoglobin, from which heme and, ultimately, high amounts of iron are released to the cerebral parenchyma [10–12]. These compounds lead to oxidative stress, blood–brain barrier disruption, and the activa- tion of different death pathways in the hematomal and perihematomal regions that lead to neuronal death [13,14]. Ferroptosis, a non-apoptotic and iron-dependent programmed cell death [15], has been described as playing a crucial role, especially at early timepoints, in the ICH model induced by collagenase [16]. Nevertheless, contrasting results have been reported regarding the ef- fect of ICH on the expression of some iron- and classical ferroptosis-related proteins [17–19]. Such discrepancies might by due, at least in part, to the severity of the model and experi- mental design/time course or the brain region analyzed. Alternatively, beyond the classical ferroptotic mechanisms directly linked to iron and loss of reactive oxygen species (ROS) detoxifying potential, the ferroptosis field is evolving and the most recently discovered ferroptotic pathways might have specific roles that have not yet been identified. To date, the whole picture of the mechanisms driving hemorrhagic stroke damage is still far from complete. Specifically, the therapeutic potential and safety profile of ATf in hemorrhagic stroke is not known, although ferroptosis, an iron-mediated type of cell death, has been previously suggested to be implicated [16]. We hypothesized that ATf, which has demonstrated benefits in ischemic stroke, could reduce the detrimental consequences of ICH and, thus, is a potential pre-hospital and pre-triage frontline treatment for stroke patients. The present work aimed to investigate (1) the effect of early treatment with ATf on the hematoma size, antioxidant status, and neurological outcome in a mouse model of ICH, and (2) the effect of ICH and ATf on the levels and expression of iron- and ferroptosis-related molecules relevant in stroke. 2. Materials and Methods 2.1. Animals Adult 9-week-old male C57BL/6J mice purchased from Charles River Laboratories were used in this study. They were housed in controlled standard conditions of temperature, humidity, and photoperiod, and had food and water ad libitum. The animal experiments were approved by the Animal Research Ethics Committee (CEEA) of the Germans Trias i Pujol Research Institute (IGTP) and the Catalan Government (references 11182 and 11131) and were conducted in a randomized manner and according to international guidelines (EU Directive 2010/63/EU) at the Comparative Medicine and Bioimage Centre of Catalonia (CMCiB). We followed the ARRIVE guidelines and were committed to the 3Rs of laboratory animal research. As postoperative care and until complete recovery, the operated animals were placed onto a heating pad and 0.8 mL subcutaneous sterile saline was injected. Euthanasia was performed by cervical dislocation after completion of the experiments. 2.2. Hypoxic/Ischemic Stroke Model (IS) The mice (n = 12) were exposed to transient unilateral brain ischemia followed by a global hypoxia procedure adapted from Guan et al. [20]. In brief, the mice were anesthetized with isoflurane (4% for induction and 1.5% for maintenance) in a 30–70% mixture of oxygen (O2 ) and nitrous oxide (N2 O) and the right common carotid artery (CCA) was transiently occluded by ligature (using a 5-0, black braided silk non-absorbable suture) (Ethicon, LLC.; San Lorenzo, Puerto Rico). The mice were allowed to recover with free access to food and water for 2 h and then placed in a hypoxia chamber at 35.5 ◦ C for 20 min, ventilated with a humidified gas mixture of 8% O2 /92% N2 at an airflow rate of 1 L/min. Then, under general anesthesia with isoflurane, the suture occluding the CCA was removed, blood flow restoration in the CCA was verified de visu, and the wound was sutured with a 4-0, black braided silk non-absorbable suture (Silkam, B.Braun; Rubí, Spain). Antioxidants 2023, 12, 1945 3 of 18 2.3. Intracerebral Hemorrhage Stroke Model (ICH) The ICH procedure was adapted from Klebe et al. [21]. Briefly, the mice were anes- thetized with isoflurane (4% for induction and 1.5% for maintenance) in a 30/70 mixture of O2 /N2 O and placed in a stereotaxic frame (Model 940 Small animal; Kopf instruments, Tujunga, CA, USA) for the intrastriatal injection of sterile bacterial collagenase type VII-S (collagenase from Clostridium histolyticum Type VII-S; Sigma-Aldrich, Saint Louis, MO, USA). Using a microinjection pump (Model UMP3T-A; Kopf Instruments, Tujunga, CA, USA) and a NanoFil 26 G syringe (World Precision Instruments; Sarasota, FL, USA), 0.06 U of collagenase in 0.4 µL sterile saline was injected into the right striatum at a rate of 200 nL/min. The coordinates from Bregma for the stereotaxic injection were 0.5 mm ante- rior, 1.7 mm lateral, and 3.0 mm ventral. After the infusion and before syringe withdrawal, the needle was left in place for 10 min and the isoflurane was decreased to 1%. The syringe was removed at a rate of 1 mm/min in order to avoid retrograde flow, and the cranial burr hole was sealed with bone wax (B.Braun Vertcare; Rubí, Spain). The animals of the sham group received the same surgical procedure and were injected with 0.4 µL vehicle. Once the common iron-related protein response in ischemic (n = 12) and hemorrhagic (n = 7) stroke models was established, the effect of hATf was tested in experimental ICH. The mice were randomly assigned to the groups ICH-vehicle (sterile saline) and ICH-human apotransferrin (hATf; Sigma-Aldrich, Saint Louis, MO, USA) at the dose of 230 mg/kg (n = 8 in each group). Exogenous hATf was used to be able to distinguish it from endogenous mouse ATf. Forty minutes after collagenase administration, vehicle or hATf was administered intravenously. 2.4. Tape Removal Test Neurobehavioral performance was tested in daily training sessions in the 3 days prior to the ICH induction, and daily for 3 days after ICH induction to assess proprioception, asymmetry, and sensorimotor impairment through the adhesive tape removal test. Briefly, a round 6 mm diameter tape was stuck in the hand palm of the mouse in the forelimb contralateral forelimb to the hemorrhagic hemisphere. Both the time the mouse spent to notice/detect and to actively remove the tape were measured in each session. The mice received training in the test before ICH induction to avoid any stress derived from the novelty of the handling associated with the test performance. 2.5. Tail Bleeding Test Nine male C57BL/6J mice were used to test a putative effect of exogenously ad- ministered hATf on coagulation (vehicle n = 5, hATf n = 4). Ten minutes after an i.v. administration of sterile saline or 230 mg/kg hATf, the tip of the tail was cut, the tail was submerged in physiological saline at 37 ◦ C to ease bleeding, and the time elapsed until the tail stopped bleeding was measured. 2.6. Blood and Brain Sampling and Processing Blood samples were obtained using EDTA-K or lithium heparin tubes (Microvette CB 300 EDTA or Microvette CB 300 lithium heparin; Sarstedt, Nümbrecht, Germany) before surgery, after the i.v. injection, and 24 h after the ICH induction. The plasma obtained after centrifugation was stored at −20 ◦ C. Three days after the treatments, the mice were euthanized via cervical dislocation, and the brains were quickly obtained and cut into 2 mm thick slices using a coronal mouse brain slicer matrix. Then, the slices were photographed on both sides. For Western blot and qPCR, the third fronto-caudal brain slice, the one showing the highest level of infarct or hematoma in the ischemic or hemorrhagic stroke models, respectively, was divided and saved separately into ipsilateral and contralateral hemispheres, flash-frozen in liquid N2 , and stored at −80 ◦ C. Further, the samples were lyophilized (Freeze Dryer; B.Braun Biotech; Melsungen, Germany) following Aliena-Valero et al.’s protocol [22] and stored at −80 ◦ C until analysis. This way, we obtained a bias-free selection of the tissue for both WB and Antioxidants 2023, 12, 1945 4 of 18 qPCR analyses. We chose this conservative procedure, despite it potentially underestimat- ing the molecular changes in the ipsilateral hemisphere due to it also containing healthy tissue, as it allows for an objective standardization of the tissue collected for analyses. For immunohistochemistry, the second rostro-caudal 2 mm thick slice obtained from the brain slicer matrix was preserved and stored at −80 ◦ C. The most caudal side of this 2 mm slice, adjacent to the tissue used for qPCR and Western blot, was sliced using a cryostat into 15 µm thick slices to be used to detect 4-HNE and NeuN. 2.7. Assessment of the Ischemic Infarct Volume in the IS Model, Hemorrhage Volume, and Brain Hemoglobin and Heme Concentration in the ICH Model The infarct volume and hemorrhage volume were quantified 72 h after treatments by measuring the 2,3,5-triphenyltetrazolium chloride (TTC)-unstained area or the hematoma area, respectively, in 2 mm thick coronal slices using ImageJ v1.53 (Wayne Rasband; NIH; Bethesda, MD, USA) and calculated as previously reported [23]. The brain hemoglobin and heme levels were measured using colorimetric detection assay kits (QuantiChrom Hemoglobin Assay Kit and QuantiChrom Heme Assay Kit; BioAssay Systems; Hayward, CA, USA) following the manufacturers’ instructions. 2.8. RT-qPCR A miRNeasy Tissue/Cells Advanced Mini Kit (Qiagen; Hilden, Germany) was used for RNA isolation. Briefly, lyophilized brain tissue samples (ipsi and contralateral hemispheres separately) were resuspended in the supplied lysis buffer with 0.01% β-mercaptoethanol and homogenized by passing the lysate through a 21 G needle at least 5 times in order to isolate the RNA. The RNA was finally recovered in RNase-free water and its concentration and purity were assessed using a NanoDrop ND-1000 Spectrophotometer (NanoDrop; Thermo Fisher Scientific; Waltham, MA, USA). The same amount of RNA was used for the reverse transcription of each sample following QuantiTect Reverse Transcription Kit (Qiagen; Hilden, Germany) instructions. The samples were first incubated with a gDNA elimination reaction for 2 min at 42 ◦ C and immediately placed on ice. The reverse transcrip- tion reaction was performed at 42 ◦ C for 15 min, followed by inactivation at 95 ◦ C for 3 min. A no-reverse transcription control was also prepared by replacing the reverse transcriptase with water. The obtained cDNA was then mixed with LightCycler 480 SYBR Green I Master (Roche Applied Science; Penzberg, Germany) and PrimeTime® qPCR Primers for different genes (Integrated DNA Technologies; Coralville, IA, USA). Real-time PCR was performed in a Real-Time PCR Roche LightCycler 480 I (Roche Applied Science; Penzberg, Germany). The reaction was initiated using a pre-incubation step of 5 min at 95 ◦ C, followed by 45 cycles of amplification at 95 ◦ C for 10 s, 60 ◦ C for 10 s, and 72 ◦ C for 10 s. Triplicates of the samples and melting curve analysis were also performed. A no-template control was included, replacing the sample cDNA with RNase-free water, as well as a positive control using Universal Mouse Reference RNA (Invitrogen; Waltham, MA, USA). Data analysis was performed using the ∆∆Ct method and GAPDH was utilized as a housekeeping gene. Commercial proprietary predesigned qPCR forward and reverse primers obtained from IDT (Integrated DNA Technologies; Coralville, IA, USA) were used (Table S1). 2.9. Western Blot (WB) Lyophilized brain samples were reconstituted in the same lysis buffer used for the hemoglobin and heme quantification. The protein content of the reconstituted samples was determined using a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific; Waltham, MA, USA). Then, 30 µg of total protein from the brain was loaded in Precast NuPAGE Midi 10% Bris-Tris (Thermo Fisher Scientific; Waltham, MA, USA). A molecular weight marker (Precision Plus ProteinTM Standards; Bio-Rad; Hercules, CA, USA) was included in the gels and GAPDH was used as a tissue sample loading control. For the TSAT assessment, 0.15 µL of plasma sample was loaded in Precast 6% TBE urea gels (U-PAGE) (Thermo Fisher Scientific; Waltham, MA, USA). These gels included Antioxidants 2023, 12, 1945 5 of 18 human apotransferrin and human holotransferrin (hHTf) (Sigma-Aldrich; Saint Louis, MO, USA), as well as in-lab prepared mouse ATf and HTf standards, which were prepared following the methods described in Byrne S et al. and Nagaoka MH et al. [24,25]. PVDF-LF membranes (Millipore; Burlington, VT, USA) were used for the electroblot- ting of the gels, which were blocked with Intercept® Blocking Buffer (Li-COR Biosciences; Lincoln, NE, USA) for 1 h. Then, the membranes were incubated overnight at 4 ◦ C with the specific primary antibodies. After that, they were incubated with NIR-conjugated secondary antibodies. The bands were measured using an Odyssey Imaging system and Image Studio Lite software v5.2 (Li-COR Biosciences; Lincoln, NE, USA). The values were measured in terms of mean integrated density (ID) in arbitrary units ((mean optical density of pixels of the specific signal) − (mean background optical density)). Then, for each protein, 1.00 was assigned to the ID of the contralateral brain hemisphere and the ID of the ipsilateral hemisphere was expressed in terms of the fold-change of that of the contralateral hemisphere. 2.10. Determination of 4-Hydroxynonenal (4-HNE) Using Immunohistochemistry (IHC) In this stage, 15 µm thick brain slices from frozen tissue were obtained using a cryostat (Leica CM1950; Leica Biosystems; Deer Park, TX, USA) on poly-L-lysine-coated glass slides and fixed in 4% paraformaldehyde. They were exposed to antigen retrieval (90–95◦ in 0.01 mol/L citrated buffer, pH 6.0, for 20 min), incubated overnight with the primary antibodies at 4 ◦ C, and then with the secondary antibodies and Hoechst (33342; Thermo Fisher Scientific; Waltham, MA, USA). Fluoromount (Sigma-Aldrich F4680; Saint Louis, MO, USA) was used as the mounting medium. Images were obtained using an AxioOberver Z1 microscope (Carl Zeiss; Oberkochen, Germany) in the Microscopy Platform Core Facility at the IGTP and analyzed using ImageJ. NeuN immunohistochemistry was macroscopically imaged on an Odyssey Imaging System. In brief, the 15 µm thick brain slice that preceded the slice used for microscopy studies was subsequently incubated with anti-NeuN antibody and with an NIR-conjugated secondary antibody for Odyssey imaging. Due to the loss of neurons, almost no NeuN staining was observed within the hematoma in the whole picture of the Odyssey-imaged brain slice. The cortical areas neighboring the hematoma had neurons potentially exposed to ICH-produced toxic compounds. Under the microscope, the areas devoid of NeuN, which are hematoma, together with the anatomical brain references easily identified in the slice under the microscope (e.g., corpus callosum, ventricles), enabled us to identify periinfarct areas of interest in the nearby cortex. Several different series of images were obtained and 4-HNE was detected and quantified in NeuN-positive neurons in the areas of interest. 2.11. TSAT Assessment Basal TSAT levels were determined before the onset of the experimental stroke proce- dures to check that mice had TSAT levels similar to those in the human population (<40%), and 24, 48, and 72 h after ICH induction. The U-PAGE gels mentioned above separate transferrin into ATf (Tf devoid of iron), monoferric Tf (mFe, an Fe atom in the C-terminal region or in the N-terminal region) and diferric Tf (diFe, holotransferrin, HTf), giving different bands according these Tf isoforms. The % TSAT was calculated using the measurement of the bands from the U-PAGE gels and the TSAT formula previously used by us and others [3,26]:   1 TSAT(%) = × mFe·Tf + diFe·Tf × 100/(ATf + mFe·Tf + diFe·Tf) 2 2.12. Antibodies The primary and secondary antibodies used for WB and IHC (see Table S2) were from Thermo Fisher Scientific (Waltham, MA, USA), Abcam (Cambridge, UK), Novus Biologicals (Centennial, CO, USA), Santa Cruz Biotechnology (Dallas, TX, USA), Green Mountain Antioxidants 2023, 12, 1945 6 of 18 Antibodies (Burlington, VT, USA), Cappel, ICN Pharmaceuticals (Costa Mesa, CA, USA), and Sigma-Aldrich (Saint Louis, MO, USA), Li-COR Bioscience (Lincoln, NE, USA) and Thermo Fisher Scientific (Waltham, MA, USA). 2.13. Statistics Original or log-transformed data were analyzed using GraphPad Prism 9. A paired or unpaired Student’s t-test or independent or repeated-measures one-way ANOVA followed by the multiple comparisons Tukey’s test were used, as appropriate. Statistical significance was considered at p < 0.05. Data are presented as the mean and SEM. 3. Results 3.1. Gross Differences and Commonalities between Ischemic and Hemorrhagic Stroke Models The nature, extent, and location of the brain regions affected by stroke differ between the two stroke models. There is a large corticostriatal infarct in ischemia, and smaller, mainly striatal, hemorrhage in ICH (Figure 1B,F). Despite this, body weight loss followed the same pattern in both ischemic and ICH mice (Figure 1E,I), and the same Antioxidants 2023, 12, x FOR PEER REVIEW applied for 7 of 20 edema (Figure 1C,G) and the cerebral midline shift induced by the ipsilateral hemisphere enlargement (Figure 1D,H). Figure Figure1.1.(A) (A)Timeline Timelinescheme schemeof the experimental of the groups experimental and procedures. groups Effect ofEffect and procedures. (B–E) ofischemic (B–E) ischemic stroke and (F–I) intracerebral hemorrhage on (B,F) infarct or hemorrhage stroke and (F–I) intracerebral hemorrhage on (B,F) infarct or hemorrhage volume, volume, (C,G) edema, (C,G) edema, (D,H) stroke-induced maximum hemispheric midline shift (broken white line) vs. the theoretical (D,H) stroke-induced maximum hemispheric midline shift (broken white line) vs. the theoretical midline (solid black line), and (E,I) body weight loss; ** p < 0.01 and *** p < 0.005 vs. pre-stroke midline (solid (repeated measures black line),ANOVA one-way and (E,I)andbody weight Tukey’s test). loss; ** prepresented Data are < 0.01 andas***thepmean < 0.005 andvs. SEM.pre-stroke (repeated Contra: measures one-way contralateral; ANOVA ipsi: ipsilateral. and Tukey’s Representative test). of images Data are represented ischemic as theinmean regions (regions whiteand SEM. in the brain Contra: slices in (B) and contralateral; ipsi:hemorrhagic ipsilateral. regions (reddish areas Representative images in the brain slicesregions of ischemic in (F) in(regions coronal in white brain slices are shown and were used to quantify ischemic infarct volume and hemorrhagic stroke in the brain slices in (B) and hemorrhagic regions (reddish areas in the brain slices in (F) in coronal volume, respectively. brain slices are shown and were used to quantify ischemic infarct volume and hemorrhagic stroke volume, respectively. 3.2. Ischemic and Hemorrhagic Stroke Share a Common Profile of Iron-Binding, Ferroptosis-Related Proteins in the Ipsilateral Brain Hemisphere 72 h after Stroke Induction Importantly, compared with the contralateral brain hemisphere, both ischemic and hemorrhagic stroke increased the protein levels of iron-storage ferritin and the iron-bind- ing and carrier transferrin in the ipsilateral hemisphere (Figure 2A,B). A modest increase Antioxidants 2023, 12, 1945 7 of 18 3.2. Ischemic and Hemorrhagic Stroke Share a Common Profile of Iron-Binding, Ferroptosis-Related Proteins in the Ipsilateral Brain Hemisphere 72 h after Stroke Induction Importantly, compared with the contralateral brain hemisphere, both ischemic and hemorrhagic stroke increased the protein levels of iron-storage ferritin and the iron-binding and carrier transferrin in the ipsilateral hemisphere (Figure 2A,B). A modest increase in the levels of divalent metal transporter 1 (DMT1), which imports ferrous iron to the cytosol, was observed in the ipsilateral hemisphere of the ischemic stroke mice only (Figure 2A); no changes were observed in the levels of transferrin receptor (TfR) (Figure 2A,B), which plays8 of 20 Antioxidants 2023, 12, x FOR PEER REVIEW a pivotal role as the main gate for iron entry into the endothelial cells of the brain capillaries and into neurons; also, TfR has recently been identified as a ferroptosis biomarker [27]. Figure Figure 2. 2. Graphs Graphsdepicting depictingthe theeffect effect of of (A)(A) ischemic ischemic stroke stroke and and (B) intracerebral (B) intracerebral hemorrhage hemorrhage on on ipsilateral brain levels of iron- and ferroptosis-related proteins 72 h after stroke ipsilateral brain levels of iron- and ferroptosis-related proteins 72 h after stroke onset, expressed as onset, expressed as fold fold change change of ofthose thoseofofthe thecontralateral contralateral hemisphere hemisphere (CLH,(CLH, mean mean represented represented by theby the dotted dotted line at line at yy == 1.0); 1.0); ** pp << 0.05, 0.05,****pp<<0.01, 0.01,*** ***p p< <0.005, 0.005, and and **** **** p