Potential role of poly (ADP-ribose) polymerase in delayed cerebral vasospasm following subarachnoid hemorrhage in rats

Abstract

Poly (ADP-ribose) polymerase (PARP) serves a key role in several neurological disorders, however, the specific role of PARP in delayed cerebral vasospasm (DCVS) following subarachnoid hemorrhage (SAH) remains unclear. The present study was conducted to clarify the possible mechanism of PARP in DCVS with the treatment of 3-aminobenzamide (3-AB), a PARP inhibitor. In the preliminary experiment, an internal carotid artery puncture SAH model, a cisterna magna double injection SAH model and prechiasmatic cistern single injection SAH model were compared with respect to mortality and neurobehavioral test results. The prechiasmatic cistern single injection SAH model was chosen to induce DCVS in the formal experiment. In the formal experiment, a total of 96 Sprague Dawley rats were randomly allocated into the sham group, the SAH group and the SAH+3-AB group and then each group was further subdivided into days 3, 5, 7 and 14 post-SAH subgroups (n=8 for each subgroup). The prechiasmatic cistern single injection SAH model was established to induce DCVS. Neurobehavioral testing and HE staining were conducted to evaluate the degree of cerebral vasospasm. PARP activity was assessed by ELISA and immunohistochemistry. An electrophoretic mobility shift assay was used to detect nuclear factor (NF)-κB DNA-binding activity. The expression of monocyte chemotactic protein 1 (MCP-1) and C-reactive protein (CRP) were measured by western blotting. Cerebral vasospasm occurred following SAH and became most severe on around day 7 post-SAH. NF-κB activity, PARP activity, the expression of MCP-1 and CRP exhibited a similar time course to cerebral vasospasm. Treatment with 3-AB alleviated the degree of cerebral vasospasm. NF-κB activity, PARP activity and the expression of MCP-1 and CRP were also suppressed by 3-AB treatment. In conclusion, PARP may serve an important role in regulating the inflammatory response and ultimately contribute to DCVS. Therefore 3-AB may be a potential therapeutic agent for DCVS.

Keywords: 3-AB; delayed cerebral vasospasm; inflammatory response; nuclear factor-κB; poly ADP-ribose polymerase; subarachnoid hemorrhage.

Figure 1.

The mortality, time course of neurological deficit score following SAH and the successful rate of inducing DCVS in different model group. (A) Mortality was calculated in the three groups and (B) neurological deficit scores were evaluated on days 3, 5, 7, 14 post-SAH. The successful rate of inducing DCVS was then calculated (C). The success rate of model formation was calculated by the proportion of rats, which were suffering typical characteristic of DCVS according to the neurobehavioral score. The IC group, the CM group and the PC group were compared. *P<0.05 vs. IC group; ^#P<0.05 vs. CM group. SAH, subarachnoid hemorrhage; DCVS, delayed cerebral vasospasm; IC, internal carotid artery puncture SAH model; CM, cisterna magna double injection SAH model; PC, prechiasmatic cistern single injection SAH model group.

Figure 2.

Mortality and macroscopic observation of rat brain following SAH. (A) The mortality was calculated in each group. (B) No obvious blood clots were observed in sham group. (C) A large amount of blood clots were observed surrounding the arteries in SAH group. SAH, subarachnoid hemorrhage; 3-AB, 3-aminobenzamide. *P<0.05 vs. the sham group

Figure 3.

The time course of neurological deficit score following SAH and the effect of 3-AB on score. The neurological deficit scores were evaluated on days 3, 5, 7, 14 post-SAH in the sham group, SAH group and SAH+3-AB group. *P<0.05 vs. the sham group, ^#P<0.05 vs. the SAH group. SAH, subarachnoid hemorrhage; 3-AB, 3-aminobenzamide.

Figure 4.

Representative morphological changes of basilar artery. The morphological changes were observed in (A) sham group, (B) SAH group on day 3, (C) SAH group on day 7, (D) SAH group on day 14, (E) SAH+3-AB group on day 3, (F) SAH+3-AB group on day 7, (G) SAH+3-AB group on day 14. The bar chart demonstrated the (H) wall thickness and (I) the internal diameter of the basilar artery in different groups. *P<0.05 vs. the sham group, ^#P<0.05 vs. the SAH group. SAH, subarachnoid hemorrhage; 3-AB, 3-aminobenzamide.

Figure 5.

Representative immunohistochemical staining of poly (ADP-ribose) on basilar artery. The immunohistochemical staining was presented in (A) the sham group, (B) SAH group on day 3, (C) SAH group on day 7, (D) SAH group on day 14, (E) SAH+3-AB group on day 3, (F) SAH+3-AB group on day 7, (G) SAH+3-AB group on day 14. The brown products demonstrated positive results of poly (ADP-ribose). (H) The bar chart demonstrated the poly (ADP-ribose) level in different groups. *P<0.05 vs. the sham group, ^#P<0.05 vs. the SAH group. SAH, subarachnoid hemorrhage; 3-AB, 3-aminobenzamide.

Figure 6.

The time course of PARP activity following SAH and the effect of 3-AB on PARP. PARP activity was detected by ELISA on days 3, 5, 7, 14 post-SAH in sham group, SAH group and SAH+3-AB group. *P<0.05 vs. the sham group, ^#P<0.05 vs. the SAH group. SAH, subarachnoid hemorrhage; 3-AB, 3-aminobenzamide.

Figure 7.

The time course of MCP-1 expression (A) and CRP expression (B) following SAH and the effect of 3-AB on MCP-1 and CRP. The expression of MCP-1 and CRP were tested by western blotting on days 3, 5, 7, 14 post-SAH in the sham group, SAH group and SAH+3-AB group. *P<0.05 vs. the sham group, ^#P<0.05 vs. the SAH group. SAH, subarachnoid hemorrhage; CRP, C-reactive protein; MCP-1, monocyte chemotactic protein-1; 3-AB, 3-aminobenzamide.

Figure 8.

The time course of NF-κB DNA-binding activity following SAH and the effect of 3-AB on NF-κB activity. NF-κB DNA-binding activity was detected by EMSA on days 3, 5, 7, 14 post-SAH in the sham group, SAH group and SAH+3-AB group. *P<0.05 vs. the sham group, ^#P<0.05 vs. the SAH group. SAH, subarachnoid hemorrhage; NF, nuclear factor.