Fig 2: Neuroprotective effect of recombinant LTF in vitro (a, b) and in a clinically relevant rat (c–e) and mouse (f, g) model of ICH. a Representative immunofluorescence of MAP2+-neurons (green) and GFAP+-astrocytes (red) in rat brain cortical neuron-glial co-cultures at 24 h after exposure to RBC lysate (an ICH-like injury in vitro), demonstrating that lysed RBCs are toxic to neurons. Arrows show the swollen soma and broken dendrites. Astrocytes are less prone to the injury. b Representative images (left part of the panel) showing density (viability index) of NeuN+-neurons (green) in the rat neuron-glial co-cultures at 24 h after the exposure to lysed RBC in the presence or absence of rLTF, (5 µg/ml), which was added to the co-culture at 30 min prior to the insult (adding lysed RBCs). The nuclei were labeled with DAPI (blue). Scale bar = 50 µm. And a bar graph of NeuN+-neuron numbers at a 10× field. Data were mean ± SEM (n = 15). *p = 0.05 from all other groups; established using one-way ANOVA followed by Newman–Keuls post-test. c–e rLTF reduces ICH-mediated brain damage in rats after ICH. Rats were treated with rLTF (5 mg/kg, i.v. at 30 min after ICH plus 2.5 mg/kg p.o., at 24 and 48 h) and assessed for c NDS (a composite score for footfault, circling, wire, postural flexing, and forward placing test) at days 1 and 3 (n = 10), d oxidative burden (4-HNE; index of lipid peroxidation) at day 3 (n = 5), and e brain edema (% of water content) at day 3 (n = 5). f, g Recombinant mouse LTF, improves outcome after ICH in mice that were treated as late as 24 h after ICH (5 mg/kg, i.v. at 24 h after ICH plus 2.5 mg/kg p.o., on day 2–7) as determined with assessing f NDS on day 1, 3, 7, and 14 (n = 11) and g iron clearance from the hematoma-affected brain on day 7 after ICH (n = 7). All data were presented as mean ± SEM. *p = 0.05, compared with the vehicle control. The p-values in d, e, g were established using paired t-test; in c, f, the longitudinal data analysis was performed and corresponding p-values were provided using the mixed effects model

Fig 3: Expression of lactoferrin (LTF) before and after cryopreservation. Western blotting analysis of protein expression in fresh and freeze-thawed ram sperm (A). Relative expression levels of LTF in fresh and frozen–thawed sperm (B). Values represent the average± standard error of the mean from three gels per group. Asterisks indicate a statistically significant difference from the fresh group: ** p<0.001 from one-way analysis of variance with Duncan’s post hoc analysis.

Fig 4: Ltf rescues neuronal ferroptosis and improved acute outcomes in hyperglycemic ICH mice. (A–G) Ltf-siRNA or Scr-siRNA was injected intraventricularly to normoglycemic ICH mice. (A, B) Immunofluorescent staining using antibodies of MDA and NeuN. White arrows indicate the MDA+NeuN+ cells (A). The MFI of MDA was quantified (B). (C, D) FJC staining was performed and the FJC+ cells were quantified. White arrows indicate FJC+ cells. (E, F) CV/Luxol fast blue staining were performed to quantify the lesion volume (black dotted bordered). (G) Neurological function and motor function were examined following ICH blindly. (H–N) Four mg/kg of rLtf was administered (i.p.) to hyperglycemic ICH mice. Immunofluorescent staining using antibodies of MDA and NeuN (H, I), FJC staining (J, K), and CV/Luxol fast blue staining (L, M) were performed. The representative images (H, J, L) and quantifications (I, K, M) are shown. Neurological function and motor function were examined following ICH blindly (N). (B, D, F, I, K, M) Student's t-test followed by Welch’s correction. (G, N) Two-way ANOVA followed by Tukey's multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001 vs corresponding Scr-siRNA (B, D, F, G) or corresponding Veh (I, K, M, N). Each group contained 6 (B, D, F, I, K, M) or 8 (G, N) animals. Scale bar: (A, H) 75 µm, (C, J) 50 µm, (E, L) 1 mm. Each experiment was repeated at least 3 times independently. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig 5: High glucose reduced neutrophilic Ltf aggravates neuronal ferroptosis in vitro. Mouse primary peripheral neutrophils were extracted and cultured in normal-glucose (NG), high-glucose (HG) medium, or transfected with siRNA before use. (A) The mRNA level of Ltf was detected by real-time RT-PCR. GAPDH was served as the internal control. (B, C) The protein levels of Ltf in neutrophils (cell lysates-Ltf) and in conditional medium (CM-Ltf) were detect using Western blot. The representative images (B) and quantifications (C) are shown. a-Tubulin was served as the internal control. (D) Schematic of co-culturing mouse primary neurons and neutrophils. (E–L) Primary mouse neurons were treated as indicated for 8 h before performing PI staining. The representative images (E, G, I, K) and quantifications (F, H, J, L) are shown. (A, C) Student's t-test followed by Welch’s correction. (F, H, J, L) One-way ANOVA followed Tukey's multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001 vs NG (A, C), Control (F, H, J), or NG-CM + Veh (L); ##p < 0.01, ###p < 0.001 vs Hemin + Veh (F, J), HG-CM + Hemin (H), or NG-CM + Hemin + IgG (L); ††p < 0.01 vs Hemin + NG-CM (F) or Hemin + Scr-siRNA (J). Each experiment was repeated 3–4 (C), 4 (A), 6 (F, J), or 3 (H, L) times independently. Scale bar: (E, G, I, K) 100 µm.