CROSS-LINKED POLYHEMOGLOBIN-SUPEROXIDE DISMUTASE-CATALASE SUPPLIES OXYGEN WITHOUT CAUSING BLOOD BRAIN BARRIER DISRUPTION OR BRAIN EDEMA IN A RAT MODEL OF TRANSIENT GLOBAL BRAIN ISCHEMIA-REPERFUSION

Figures & data

Figure 1. Molecular distribution of cross-linked and non–cross-linked ultrapure bovine SF-Hb. Elution profiles of 5-, 10-, 24-hour cross-linked PolyHb, PolyHb-SOD-CAT and non–cross-linked bovine SF-Hb were obtained by running 1 ml of the 10× diluted samples on Sephadex G-200 1.6 cm×70 cm column (VT=105.5 ml, Vo=38 ml, equilibrated with 0.1 M Tris-HCl, pH 7.5, and eluted at 11.5 ml/hr).

Table 1. Molecular Components of PolyHb and PolyHb-SOD-CAT

Cross-Linking Time	Samples	<100 kDa	100 to 450 kDa	>450 kDa
		Component Percentage
Before ultrafiltration
5 hours	PolyHb	26	25	49
	PolyHb-SOD-CAT	26	25	49
10 hours	PolyHb	19	24	57
	PolyHb-SOD-CAT	19	24	57
24 hours	PolyHb	10	18	72
	PolyHb-SOD-CAT	10	18	72
After ultrafiltration
5 hours	PolyHb	21	24	55
	PolyHb-SOD-CAT	21	24	55
10 hours	PolyHb	14	23	63
	PolyHb-SOD-CAT	14	23	63
24 hours	PolyHb	7	16	77
	PolyHb-SOD-CAT	7	16	77

Table 2. Molecular Distribution and SOD and CAT Activity

Molecular Wt.	MW Component (%)	SOD Activity (%)	Catalase Activity (%)
PolyHb-SOD-CAT sample
>450 kDa	77	83±3	82±2
100 to 450 kDa	16	7±2	11±1
<100 kDa	7	4±1	5±1
PolyHb sample
>450 kDa	77	0	0
100 to 450 kDa	16	0	0
<100 kDa	7	0	0

Figure 2. Oxygen-hemoglobin dissociation. The 24 hours of cross-linking with glutaraldehyde did not significantly affect the P50 value of SF-Hb. PolyHb and PolyHb-SOD-CAT P50 values were 26.1±0.2 mmHg and 24.7±0.2 mmHg, respectively. The SF-Hb P50 value was 27.8±0.0 mmHg. All oxygen-hemoglobin dissociation curves are presented as averages of 6 trials (n=6).

Figure 3. Determining the required ischemic duration. Animals were subjected to either 0, 20, 30, 40, or 60 minutes of ischemia before reperfusion. With 60 minutes of ischemia, the level of Evans blue in the sham control was significantly lower than that of oxygenated saline, stroma-free Hb (SF-Hb), and PolyHb. Statistical significance is P<0.01.

Figure 4. Evans blue extravasation. PolyHb-SOD-CAT significantly attenuated the severity of BBB disruption as compared to saline, SF-Hb, SF-Hb+SOD+CAT, and PolyHb. Statistical significance is P<0.01.

Figure 5. Brain edema: changes in brain water content. The changes in brain water content of PolyHb-SOD-CAT–treated animals were not significantly different from that of the sham control. The increase in water contents of saline, SF-Hb, SF-Hb+SOD+CAT, and PolyHb were significantly different from that of the sham control and PolyHb-SOD-CAT group by the 4th hour and increased with time. Statistical significance is P<0.01.