Hypohaptoglobinemia (HHp) aggravates ICH-mediated damage.
(A) Photograph of representative rat brain coronal section illustrating location of hematoma produced with intrastriatal injection of lysed blood (M-ICH). (B) Representative photomicrograph of H&E stained cryosection showing edema surrounding hematoma (outlined with dotted lines), as captured at 24h after M-ICH. (C) Bar graph illustrating the densitometrical analysis of immunoreactivity (immuno-dot blots technique) for 3’-NT and 4-HNE in the hematoma-affected tissue homogenates of normohaptoglobinemic control (ICH) and HHp (ICH+HHp) rats 24h after M-ICH. The data are expressed as mean ± SEM (n=5). *p≤0.05, from the control (ICH). (D) Bar graph illustrating brain edema measured by wet-to-dry weight ratio (% of water) at 24h after M-ICH in the control (ICH) and HHp (ICH+HHp) rats. The data are expressed as mean±SEM (n=7). *p≤0.05, from the control (ICH)

Hp in the hemorrhage-affected striatum is increased after ICH and is augmented by SF. The SD rats were injected intraperitoneally with either saline (ICH) or SF (ICH+SF; 5 mg/kg) at 30 min and 24h after ICH. (A) Photograph of RT-PCR products for Hp on representative agarose gel, and bar graph illustrating the densitometrical quantification of Hp mRNA at 3h, 1d, 2d, 3d and 7d after ICH. The data are calculated as mean±SEM (n=4) and expressed as fold increase over the naïve groups. *p<0.05, from naïve and the saline control at the same time point; **p<0.05, from naive. (B) Photograph of the representative immunoblot for Hp and bar graph illustrating the densitometrical quantification of Hp protein at 3h, 1d, 2d, 3d and 7d after ICH in rats. The data are calculated as mean±SEM (n=4) and expressed as fold increase over the naïve groups. *p<0.05, from naïve and ICH control at the same time point; **p<0.05, from naive. (C) Table indicating blood plasma Hp level in naïve mice (first column) and in mice with (ICH+SF) and without (ICH) treatment with SF as determined at 1, 2 and 3 days after ICH. The data are expressed as mean±SEM (n=5). *p ≤0.05, from naive; **p<0.05, from naive and ICH at the same time point.

(A) Bar graph of LDH released into culture media by neurons in culture exposed to 0.1~50 µM Hb for 24h. The data are expressed as percentage over the media control and displayed as mean±SEM (n=3). *p≤0.05, from the media control. (B) Photomicrograph of MAP2 immunofluorescence in the neuronal cultures after exposure to Hb, demonstrating morphological damage. The 15 day-old neurons in culture were incubated with: (a) 3 µM Hb or (b) the complexes of Hp-Hb (4.5 µM Hp and 3.0 µM Hb were co-incubated for 15min prior to the use) for 24h and the morphological integrity was assesses using immunostaining for MAP2. The arrows show the breaks of neurites. (C) Photograph of the immunoblot analyzing NF-M in lysates of neurons in culture subjected to Hb alone or as complexes with Hp (Hb+Hp), under experimental conditions described for the above panel B. (D) Bar graph of LDH released into culture media by neurons in response to: saline (Con), 3µM Hb, 4.5µM Hp, Hp+Hb, 0.5mM NMDA, 0.5mM NMDA+ 4.5µM Hp, or 0.5mM NMDA+10µM MK-801 (MK). The values of LDH are expressed as percentage change over the control (Con). The data are displayed as mean ± SEM (n=6). *p≤0.05, from Con, Hp and Hb-Hp; **p≤0.05, from NMDA and NMDA+Hp.

(A) Bar graph illustrating LDH, release by 15d-old mouse neurons in culture subjected to “ICH-like” injury (lysed RBC + hypoxia) in presence of conditioned media from OEC prepared from either Hp-KO or Hp-Tg mice or blank (control) media. The conditioned or blank media was transferred into the neuronal culture (replacing 2/3 volume of the media used to grow neurons) and incubated for 15min before induction of “ICH-like” injury. The cytotoxicity level after media transfer (before injury; 0h) and at 24h with and without induction of injury was assessed by the analysis of LDH released into the culture media. The LDH release is expressed as optical density (OD). The data are calculated as mean±SEM (n=3). * p≤0.05, from Hp-KO. (B) Bar graph of LDH release by Hp-deficient (Hp-KO) and Hp-overexpressing (Hp-Tg) OEC upon injury induced by exposure to RBC-lysates. The levels of LDH released into culture media during first 16h after induction of the insult are expressed as fold increase over the control (without RBC lysate). The data are calculated as mean±SEM (n=6). *p≤0.05, from Hp-KO. (C) Representative photomicrograph of OEC from Hp-KO or Hp-Tg mice that were exposed to RBC lysates for 16h. Oligodendroglia were visualized by immunostaining for MBP. Note, the oligodendrocytes overexpressing Hp (Hp-Tg) show better-preserved morphology, as compared to Hp-deficient (Hp-KO) ones. Scale bar = 50 µm. (D) Representative Western blot for MBP as determined in homogenates from OEC exposed to 1µM or 3µM Hb in presence or absence of 4.5µM Hp for 24h. *p≤0.05, from Control (Cont).

Hp protects brain from injury produced by M-ICH.
(A) Bar graph illustrating the composite neurological deficit scores (NDS; left panel) and deficit scores for the individual (postural reflex, corner, right forelimb pacing and right footfault) tests at 7 days after M-ICH in wild-type (WT), Hp-KO and Hp-Tg mice. The data are expressed as mean±SEM (n=9). *p≤0.05, from all the remaining groups. (B) Photograph of immunoblot showing bands of NF-M, Tau and MBP in hematoma-affected striatum of WT, Hp-KO and Hp-Tg mice with (+) and without (−) M-ICH injury at 7d after the insult. (C) Bar graph of densitometrical quantification of NF-M, Tau and MBP on the immunoblots. The data are calculated as percentage over naïve animals and expressed as mean±SEM (n=5). *p≤0.05, from all the remaining groups.

The representative Hp expression pattern in the ICH-affected brain after ICH. The rats at 24h after M-ICH were analyzed for presence of Hp using immunohistochemistry. Hp immunopositive cells (green) were detected in corpus callosum and in the basal ganglia around the hematoma. The double immunofluorescence labeling with anti-myelin basic protein (MBP; red) and anti-Hp (green) antibodies indicates that Hp co-localizes with oligodendrocytes (MBP immunopositive cells). Scale bar = 50 µm. “ICH”--- hematoma.

Hp is expressed by oligodendroglia and its expression is stimulated by SF.
(A). Photomicrograph of myelin basic protein (MBP)-immunolabeled oligodendrocyte expressing Hp. The mixed neuronal-glial co-cultures were incubated with 1 µM SF for 24h and than immunolabeled for MBP (a; green) and Hp (b, red). (c).Merged image of MBP and Hp. The nuclei were visualized with Hoechst 33258 (blue, c). Note that among all cells in the field, only MBP-positive cell is immunopositive for Hp. Hp proteins is detected in the soma and fine processes of MBP-positive cells. Scale bar = 100 µm. (B) Photograph of RT-PCR product for Hp on representative agarose gel (mRNA upper panel) and Hp immunoblot (protein, lower panel), illustrating levels of Hp expression in the oligodendroglia-enriched cultures in absence (CON) or presence of sulforaphane (SF; 1 µM for 24h). (C) Bar graph quantifying Hp protein in the oligodendrocyte-enriched culture media, at 48h after treatment with 0.1~2 µM SF, determined by ELISA. *p<0.05, from vehicle control.