Microvascular and systemic responses to novel PEGylated carboxyhaemoglobin-based oxygen carrier in a rat model of vaso-occlusive crisis

Figures & data

Table 1. Clinical characteristics.

Variable	Normal	HbSS
Number of donors	N = 2	N = 16
Donor age (y)	25 ± 2.0	38 ± 2.7
Gender
Male	1	0
Female	1	16
Medications
Barbiturates	0	1
Opioids	0	15
Immunosuppressives	0	1
NSAIDs	0	6
Hydroxyurea treatment	0	0
Recent blood transfusion	0	2
In VOC at time of collection	0	5

Data were included based on relevance to the pathology of sickle cell disease. A specific point of interest is the common usage of opioids for pain management of episodic vaso-occlusive crisis in nearly all affected patients. Age data are presented in mean years ± SEM. All other data represents number of patients for which the cell applies.

Figure 1. Microvascular Panel. Images were captured by a colour digital camera and adjusted for brightness and contrast to account for differential thicknesses in spinotrapezius muscles and minor shifts in focus. (A) Images were captured at baseline, immediately following ET and immediately after treatment. Each series is taken from a single site in an animal representative of its respective group. VOC is associated with a decrease in perfused vessels and a “blue shift” of larger vessels. Treatment with PEG-COHb but not LRS causes “red shift,” which is associated with a return of oxygenated haemoglobin to those blood vessels. (B) Each set of images is from a different experiment. The 5X image shows a VOC aggregate – denoted by purple arrow – that shifts position during treatment and then dissolves at the completion of treatment. The 40X image shows this effect in a smaller vessel where the aggregate can be seen undergoing a back-ended dissolution. Direction of flow is right to left in these images.

Figure 2. Tissue interstitial oxygenation. Tissue oxygenation was measured in the interstitial fluid bathing the capillary beds of the Distal, Medial and Proximal regions of the spinotrapezius muscle as the photograph diagrams. The Distal region was more sensitive to initial VOC aggregate formation and thus first developed local hypoxia. Other regions were not driven to hypoxia, but still showed reductions in P_ISFO₂ following ET and responses to fluid treatment. P_ISFO₂ was normalized to baseline for better visual comparisons and only intergroup differences are illustrated since tissues remained below baseline levels following ET. N = 8 per group. *p<.05, **p<.01, ***p<.001 and ^****p<.0001.

Table 2. Systemic variables.

Measurement	BL	ET0	PT0	PT60	PT120	PT180	PT240
Sham
MAP	122 ± 4.4	48 ± 5.1***	71 ± 6.9***	89 ± 13.2^**,Ϯ	98 ± 15.0	119 ± 7.0
PP	46 ± 3.6	29 ± 4.0	51 ± 5.3	39 ± 6.9^Ϯ	42 ± 7.2	49 ± 7.5
HR	452 ± 13.7	443 ± 17.7	455 ± 17.4	495 ± 9.2	522 ± 15.2*	569 ± 11.0**
Temp	36.0 ± 0.19	35.7 ± 0.34	35.7 ± 0.39	36.0 ± 0.44	36.9 ± 0.04	36.2 ± 0.23
LRS
MAP	129 ± 4.8	50 ± 5.3***	92 ± 7.6**	116 ± 4.9β	86 ± 17.7**	111 ± 23.0
PP	45 ± 1.6	31 ± 6.5	62 ± 7.9	61 ± 4.8β	50 ± 6.4	71 ± 2.5
HR	455 ± 9.6	421 ± 11.0	444 ± 9.2	486 ± 14.8	487 ± 11.2	522 ± 12.0
Temp	36.0 ± 0.19	35.6 ± 0.21	35.6 ± 0.17	36.5 ± 0.22	36.0 ± 0.36	36.2 ± 0.20
PEG-COHb
MAP	120 ± 8.1	46 ± 4.0***	106 ± 3.0βββ	121 ± 4.0ββ	124 ± 4.0^ϮϮ	113 ± 5.6	120 ± 7.0
PP	45 ± 1.5	32 ± 4.7	63 ± 4.2	61 ± 5.8β	61 ± 4.3	57 ± 4.5	51 ± 14.4
HR	441 ± 15.1	410 ± 18.8	417 ± 12.6	437 ± 11.8ββ^Ϯ	469 ± 9.5β	486 ± 7.2β	506 ± 8.2
Temp	35.9 ± 0.15	35.8 ± 0.16	35.8 ± 0.20	36.8 ± 0.20	36.8 ± 0.19	36.7 ± 0.12	36.9 ± 0.17

MAP: mean arterial pressure (mmHg); PP: pulse pressure (mmHg); HR: heart rate (BPM); Temp: temperature (°C). Systemic variables for MAP, PP, HR and Temp are presented here as mean ± SEM. Two-way ANOVA with Tukey’s HSD post-hoc was performed to detect intra- and inter-group differences. Statistical comparisons within groups are compared to baseline (BL). N = 8 per group. ^{*, **, ***} indicate p<.05, .01 and .0001 compared to BL, respectively. ^{β, ββ, βββ} indicate p<.05, .01, .001 and .0001 for inter-group comparison to Sham, respectively. ^{Ϯ, ϮϮ} indicate p<.05 and .01 for inter-group comparison to LRS, respectively.

Figure 3. Post-treatment survival. Study groups were assessed for duration of survival from the end of treatment. PEG-COHB showed a significant increase in survival versus LRS and Sham. *** Indicates p<.001 result from overall Log-rank Mantel–Cox test between both LRS and Sham versus PEG-COHB groups. N = 8 per group.

Figure 4. Lactate. Blood samples were collected at BL, ET₀ and the experiment’s end (defined as 4 h post-treatment or a mean arterial pressure of <50 mmHg in demise animals) and assessed for serum lactate as an indicator of systemic metabolism. Induction of VOC produced a significant (not illustrated) increase in lactate across groups. Treatment produced a separation by the end of experiment with PEG-COHb showing no change versus ET₀ (not illustrated). Intergroup comparisons are shown. N = 8 per group. ^****p<.0001.