The Effects And Mechanisms Of Deferoxamine On Cerebral Autoregulation After Intracranial Hemorrhage

Background and Objective:Intracerebral hemorrhage(ICH)is the most common type of hemorrhagic stroke,accounting for 27.9% of new strokes worldwide in 2019,and characterized by high disability and mortality.It has been reported that impaired cerebral autoregulation(CA)following ICH is independently associated with poor prognosis of patients,suggesting that improvement of CA could be a promising target for ICH treatment.Deferoxamine(DFO),a hydrophilic iron chelator,has been demonstrated to alleviate neuroinflammation,neuronal death and brain edema and improves neurological deficits,etc.in different ICH models.DFO infusion is associated with cerebral vasodilation.Moreover,DFO improves cerebrovascular functions in the elderly,which may be related to the upregulation of hypoxia-inducible factor-1α(HIF-1α)signaling pathway.Based on the above background,this study focused on the dynamic changes of CA after ICH,investigated whether DFO could improve ICH-induced CA dysfunction,and further explore whether HIF-1α mediates the improvement effect of DFO,providing new theoretical evidence for the application of DFO in ICH treatment.Methods:This study was mainly divided into 3 parts.Part 1 aims to clarify the time characteristics of the upper and lower limits of the static cerebral autoregulation after ICH.Rat ICH models were induced by injection of collagenase using a brain stereotactic instrument.At day 1,3,7,and 14 after ICH,femoral artery catheterization was performed on ICH rats and mean arterial pressure(MAP)was monitored by a multi-channel physiological instrument,simultaneous monitoring of ipsilateral cerebral blood flow(CBF)perfusion using a doppler flow meter.MAP was regulated by bloodletting or phenylephrine injection,and CA was assessed by recording the change in CBF when MAP changes.Part 2 was designed to clarify the therapeutic effect of DFO on the impaired CA after ICH.DFO(50 mg/kg)was injected intraperitoneally within 2 h after ICH onset,twice per day for a maximum of 14 days,with an equal volume of saline as a control.Detects MAP and CBF and plots CA curves.Western blot,q PCR and immunofluorescence staining were used to analyze the changes of HIF-1α,VEGF,EPO,eNOS and p-eNOS expression in tissues around the hematoma.Part 3 was designed to investigate the role of HIF-1α in the regulation of CA by DFO.The HIF-1α inhibitor PX-478(20 mg/kg)was administered intraperitoneally once daily for 3days before molding,while the control group received an equal volume of saline.Draw CA curves and analyze the changes in HIF-1,VEGF,EPO,eNOS,and p-eNOS expression around hematoma.In addition,immunofluorescence staining of TUNEL,GFAP,and Iba1 were also used to assess neuronal death and inflammatory response.Results:1.Compared with Sham group,there was no significant change in the upper limit of CA in ICH rats;the lower limit of CA was impaired at day 1 after ICH(P<0.01),peaked at day 3 after ICH(P<0.01),and gradually recovered at day 14 after ICH(P<0.05).2.Compared with the ICH group,the CA curve of day 1 of DFO injection showed an upward trend,but the difference was not significant.The CA curve at day 1 of DFO injection had an upward trend compared with the ICH group,but the difference was statistically significant.In contrast,the CA curve at day 3 of DFO injection was significantly shifted upward in the range of MAP 30-80 mm Hg,which significantly reduced the percentage change in CBF at the time of MAP drop(P<0.01).Compared with the ICH group,the CA curve at day 7 and 14 of DFO injection still showed an upward shift at lower MAP ranges,and this upward shift was significant at 30,35,and45 mm Hg on treatment day 7 and at 35,40,and 60 mm Hg on treatment day 14.3.Compared with the Sham group,the expression of HIF-1α,VEGF,and EPO in the tissue around the hematoma was significantly higher in the ICH 3d group(P<0.001,P<0.01,P<0.05);compared with the ICH 3d group,DFO injection further increased the expression of HIF-1α,VEGF,and EPO(P<0.001,P<0.05,P< 0.01).In addition,ICH also altered the expression of cerebral regulation-related proteins such as eNOS and p-eNOS.Western blot showed that eNOS expression was elevated in the ICH 3d group compared with the Sham group(P<0.001),while p-eNOS expression was significantly decreased(P<0.01);compared with the ICH 3d group,expression of eNOS and p-eNOS was significantly higher in the ICH+DFO 3d group(P<0.05、P<0.001).Immunofluorescence staining further supported the Western blot results.4.Compared with the ICH+DFO 3d group,PX-478 injection decreased the improvement of DFO on CA dysfunction after ICH and significantly reduced the levels of HIF-1α,VEGF,EPO,eNOS and p-eNOS in the ipsilateral brain(P < 0.001).In addition,ICH resulted in increased TUNEL staining positive cells,microglia counts and GFAP fluorescence intensity compared to the Sham group(P < 0.001),and DFO attenuated above changes(P < 0.001).Additionally,the ICH+DFO+PX-478 3d group showed a higher percentage of TUNEL-positive cells than the ICH+DFO 3d group(P<0.05),indicating that neuroprotective effects induced by DFO were diminished.However,there was no statistically significant difference in the fluorescence intensity of GFAP or microglia count.Conclusions:1.DFO injection improves the impaired CA following ICH.2.The mechanism of DFO improving the CA in ICH rats may be implemented by up-regulation of the HIF-1α signaling pathway.