Subarachnoid hemorrhage in rats: effect of singular or sustained hemodilution with alpha-alpha diaspirin crosslinked hemoglobin on cerebral hypoperfusion.

OBJECTIVE: To evaluate the effect of singular or sustained hemodilution, with alpha-alpha diaspirin crosslinked hemoglobin (DCLHb), on the area of hypoperfusion after subarachnoid hemorrhage. DESIGN: Prospective animal study. SETTING: Animal research laboratory. SUBJECTS: Isoflurane anesthetized, mechanically ventilated rats. INTERVENTIONS: Subarachnoid hemorrhage was induced by injecting 0.3 mL of blood into the cisterna magna. The animals were randomly assigned to one of the following groups (n = 16 in each hemodilution group; eight animals received a single treatment of hemodilution after subarachnoid hemorrhage; and, for eight animals, treatment was sustained for 48 hrs): control group (n = 8), no hematocrit (45%) manipulation; DCLHb group (n = 16), hematocrit decreased to 30% with DCLHb; or Alb group (n = 16), hematocrit decreased to 30% with human serum albumin. After 48 hrs, the area of hypoperfusion (cerebral blood flow < 40 ml/100g/min) was determined with 14C-iodoantipyrine in five coronal brain sections. MEASUREMENTS AND MAIN RESULTS: For both singular and sustained treatment, the area of hypoperfusion was less in both hemodilution groups than in the control group (p<.05). For four of the five coronal brain sections, no differences were found between the DCLHb and Alb groups within a given hemodilution protocol. In addition, in four of the five coronal brain sections for the DCLHb hemodilution groups and in all five sections for the albumin hemodilution groups, the area of hypoperfusion was less for rats that received sustained hemodilution compared with their respective groups in the singular treatment protocol (p<.05). CONCLUSIONS: These data support the hypothesis that hemodilution with molecular hemoglobin decreases hypoperfusion after subarachnoid hemorrhage and that sustained hemodilution is more effective than singular treatment. The data do not support the notion that intravascular DCLHb has an adverse effect on cerebral ischemia after subarachnoid hemorrhage.

Alpha-alpha diaspirin crosslinked hemoglobin, nitric oxide, and cerebral ischemic injury in rats.

Prior studies indicate that alpha-alpha diaspirin crosslinked hemoglobin (DCLHb) decreases cerebral ischemia. One mechanism whereby DCLHb may ameliorate cerebral ischemia is by binding nitric oxide (NO), which has been implicated as neurotoxic. We assessed the effect of L-NAME (NO synthase inhibitor) and L-arginine (NO substrate) on ischemic brain injury after DCLHb infusion. Rats were randomized to one of the following groups: Control-no hematocrit manipulation; DCLHb-hematocrit decreased to 16% with 10% DCLHb; DCLHb/L-NAME-hematocrit decreased to 16% with DCLHb, and L-NAME given; DCLHb/L-arg-hematocrit decreased to 16% with DCLHb, and L-arginine given. After 90-min of middle cerebral artery occlusion and 4-hr of reperfusion, infarct volume was determined with TTC stain. Infarct volume (mm3, mean +/- SD) was greater in the Control group (142 +/- 16) than the DCLHb (43 +/- 12), DCLHb/L-NAME (45 +/- 14), and DCLHb/L-arg (71 +/- 18) groups (p < 0.05); was greater in the DCLHb/L-arg group than the DCLHb and DCLHb/L-NAME groups (p < 0.05); but was not different between the DCLHb and DCLHb/L-NAME groups. These data indicate that DCLHb decreases ischemic brain injury, and that binding NO may be one mechanism by which DCLHb decreases ischemic brain injury.

Experimental subarachnoid hemorrhage in rats: effect of intravenous alpha-alpha diaspirin crosslinked hemoglobin on hypoperfusion and neuronal death.

BACKGROUND: Hemodilution with diaspirin crosslinked hemoglobin (DCLHb) ameliorates occlusive cerebral ischemia. However, subarachnoid hemoglobin has been implicated as a cause of cerebral hypoperfusion. The effect of intravenous DCLHb on cerebral perfusion and neuronal death after experimental subarachnoid hemorrhage was evaluated. METHODS: Rats (n = 48) were anesthetized with isoflurane and subarachnoid hemorrhage was induced by injecting 0.3 ml of autologous blood into the cistema magna. Each animal received one of the following regimens: Control, no hematocrit manipulation; DCLHb, hematocrit concentration decreased to 30% with DCLHb; or Alb, hematocrit concentration decreased to 30% with human serum albumin. The experiments had two parts, A and B. In part A, after 20 min, cerebral blood flow (CBF) was assessed with 14C-iodoantipyrine autoradiography. In part B, after 96 h, in separate animals, the number of dead neurons was determined in predetermined coronal sections by hematoxylin and eosin staining. RESULTS: Cerebral blood flow was greater for the DCLHb group than for the control group; and CBF was greater for the Alb group than the other two groups (P < 0.05). In one section, CBF was 45.5 +/- 10.9 ml x 100 g(-1) x min(-1) (mean +/- SD) for the control group, 95.3 +/- 16.6 ml x 100 g(-1) x min(-1) for the DCLHb group, and 138.1 +/- 18.7 ml x 100 g(-1) x min(-1) for the Alb group. The number of dead neurons was less in the Alb group (611 +/- 84) than in the control group (1,097 +/- 211), and was less in the DCLHb group (305 +/- 38) than in the other two groups (P < 0.05). CONCLUSIONS: These data support a hypothesis that hemodilution decreases hypoperfusion and neuronal death after subarachnoid hemorrhage. The data do not support the notion that intravascular molecular hemoglobin has an adverse effect on brain injury after subarachnoid hemorrhage.

Effect of oncotic pressure of diaspirin cross-linked hemoglobin (DCLHb) on brain injury after temporary focal cerebral ischemia in rats.

Previous studies have shown that diaspirin cross-linked hemoglobin (DCLHb, 10 g/dL) decreases cerebral ischemia and the resultant injury in a dose-dependent manner, requiring large volumes of DCLHb for maximum efficacy. We assessed the effect of a more concentrated (20 g/dL) and more hyperoncotic preparation of DCLHb on cerebral infarction volume. Immediately after middle cerebral artery occlusion, rats were randomized to one of the following groups: Control, hematocrit not manipulated; 10/Hb, hematocrit decreased to 30% with 10% DCLHb (oncotic pressure 43 mm Hg); 7.5/Alb, hematocrit decreased to 30% with 7.5% albumin (oncotic pressure 43 mm Hg); 20/Hb, the same dose of DCLHb (20%, oncotic pressure 129 mm Hg) as the 10/HB group (half the volume); or 15/Alb, the same dose of albumin (15%, oncotic pressure 130 mm Hg) as the 7.5/Alb group half the volume). After 90 min of ischemia, 72 h of reperfusion was allowed. Infarction volume (mm3, mean +/- sd) was less in the DCLHb groups (10/Hb = 79 +/- 17; 20/HB = 51 +/- 14) than the oncotically matched albumin groups (7.5/Alb = 124 +/- 21; 15/Alb = 85 +/- 18) and the Control group (135 +/- 17) (P < 0.05). These data indicate that in this model of cerebral ischemia, DCLHb decreases ischemic brain injury more effectively than albumin, and that a hyperoncotic preparation of DCLHb is preferable.