Experimental Study The Neuroprotective Role Of Progesterone On Early Brain Injury In Male Rats After Subarachnoid Hemorrhage

PartⅠ: The Neuroprotective effects of progesterone on early brain injury in male rats after subarachnoid hemorrhageObjective: To investigate the progesterone treatment whether significantly ameliorated the EBI, such as the clinical behavior scale, brain edema, and blood-brain barrier (BBB) impairment.Methods:Sixty health male Sprague-dawley(SD) rats were assigned randomly into following groups: Control group(n=12), SAH group(n=12), SAH + progesterone group.The SAH + progesterone group then was assigned into three subgroups according to the dose of progesterone (4mg/kg/d,16mg/kg/d,32mg/kg/d: n=12). All SAH animals were subjected to injection of 0.3 ml fresh arterial, non-heparinized blood into prechiasmatic cistern in 20 seconds. Male rats were given 4 / 16 /32mg/kg injections of progesterone at post-SAH hours 1, 6, and 24. Brain samples adjacent to the clotted blood were extracted at 48 h after SAH. Control animals underwent exactly the same procedure as described above with the exception that no blood was injected intracisternally. The clinical behaviors function after experimental SAH were observed; cerebral edema and blood-brain barrier permeability were detected at 48h after experimental SAH.Results: Impact of Progesterone on Clinical Behavior Function after Experimental SAH. As compared with control group, clinical behavior function impairment caused by SAH was evident in SAH subjects (P < 0.01). No significant difference was seen between the SAH group and 4mg/kg/d and 32mg/kg/d progesterone group (P >0.05). The 16mg/kg/d progesterone treated rats showed better performance in this scale system than SAH group rats at 24 h after SAH and the difference was statistically significant (P <0.01).Progesterone-Ameliorated Cerebral edema after Experimental SAH. Significant increase (P <0 .05) in water content was detected in the brain samples at 48 h after SAH when compared with rats in control group. The mean value of brain water content in the cortex was decreased by 16mg/kg/d progesterone administration (P < 0.05) as compared with SAH group and 4 or 32mg/kg/d group. The results suggested that moderae progesterone treatment could attenuate brain edema in this rat SAH model. Influence of progesterone on the Blood-Brain Barrier Permeability Following SAH. Rats in SAH group demonstrated a significant increase (P <0 .01) in BBB permeability to Evans blue relative to rats of control group. Administration of moderate progesterone significantly inhibited Evans blue extravasation (P < 0.05), indicating a reduced BBB opening in response to progesterone treatment.Conclusions: 1. As compared with control group, clinical behavior function impairment caused by SAH was evident in SAH subjects. SAH group significantly attenuate blood-brain barrier (BBB) , increase brain edema compared to control group. 2. Post-SAH progesterone treatment significantly ameliorated the EBI, such as the clinical behavior scale, brain edema, and blood-brain barrier (BBB) impairment. It suggests that.Part II: Progesterone administration modulates cortical TLR4/NF-κB signaling pathway after subarachnoid hemorrhage in male ratsObjective: The aim of the current study was to investigate whether progesterone administration modulated TLR4/NF-κB pathway signaling pathway in the brain at the early stage (EBI) of SAH.Methods: Twenty-four male Sprague Dawley rats weighing from 280 to 350 g were randomly divided into control group(n=6), SAH group(n=6), SAH +vehicle group(n=6) and SAH + progesterone group(n=6) .All SAH animals were subjected to injection of 0.3 ml fresh arterial, non-heparinized blood into prechiasmatic cistern in 20 seconds. Male rats were given 0 or 16 mg/kg injections of progesterone at post-SAH hours 1, 6, and 24. Brain samples(adjacent to the clotted blood) were extracted at 48 h after SAH. Control animals underwent exactly the same procedure as described above with the exception that no blood was injected intracisternally.We measured the expressions of TLR4, NF-κB, intercellular adhesion molecule-1 (ICAM-1), and monocyte chemoattractant protein-1 (MCP-1) by western blot;and TLR4, NF-κB, and MCP-1 expressions by immunohistochemistry.Results: Western Blot Analysis for Detecting TLR4, NF-κB, ICAM-1, and MCP-1 Expressions after SAH. These proteins were expressed at a low level in the rat brains of control group. The levels of TLR4, NF-κB, ICAM-1, and MCP-1 were significantly increased in the cortex in SAH group as compared with that of sham-operated groups (P < .01). The protein expressions had no significant difference between SAH group and SAH + vehicle group (P > .05).The expressions of TLR4, NF-κB, ICAM-1, and MCP-1 in the brains of SAH + progesterone group were significantly lower than those of the SAH + vehicle group.Immunohistochemistry for TLR4, NF-κB, and MCP-1 Expressions after SAH. To assess the localization of TLR4/NF-κB pathway, immunohistochemistry for TLR4,NF-κB, and MCP-1 was performed. A few TLR4, NF-κB, or MCP-1 positive cells were observed in the control group, which indicates the constitutional activity of TLR4/NF-κB pathway in the control brain of rats. Increased positive cells in the SAH groups could be found in the brain samples. These proteins'immunoreactivity was mainly present in neurons and a little in glia cells .In SAH + progesterone group, the number of positive cells was decreased.Conclusions: SAH could induce an activation of TLR4/NF-κB signaling pathway that might play a central role in the inflammatory response that leads to secondary insults after SAH. The therapeutic benefit of post-SAH progesterone administration might be due to its salutary effect on modulating TLR4/NF-κB signaling pathway.