| Intracerebral hemorrhage(ICH)accounts for the debilitating brain damage in 10-15% of all stroke patients and is the cause of morbidityand high rate of mortality 30-50%.Currently the only proven treatments for ICH are surgical decompression of large,life-threatening hematomas and other supportive strategies.Hematoma-induced primary injury consists of physical disruption and mass effect to the brain's cellular architecture.Subsequently,the release of hemorrhagic components,brain-derived damage signals,activation of the coagulation cascade and the complement system trigger the formation of intracerebral inflammation leading to secondary brain injury.Microglia areactivated,followed by infiltration of neutrophils,monocytes,and lymphocytes,thus exacerbating brain injury around the hematoma via release of reactive oxygen species,inflammatory mediators,and direct cytotoxic effects.Breakdown of the blood-brain barrier(BBB)facilitates the infiltration of leukocytes and can,itself,cause inflammation.As a result,this post-ICH inflammatory cascade contributes to the formation of edema that surrounds the hematoma,exacerbates the mass effect,and amplifies the cell death process.Perihematomal edema(PHE)increases in volume by about 75% in the first 24 hours after an ICH,peaks about 5 to 6 days later,and lasts up to 14 days.The extent of PHE is considered an important predictor for the outcome of ICH.In a pilot study,oral administration of the S1 PR modulator fingolimod attenuated PHE progression,reduced the MMP-9 level in plasma and improved the clinical outcomes of ICH patients.Infection is an important clinical complication after stroke,contributing to increased morbidity and mortality.The mechanisms governing the increased incidenceof infection after ICH are unclear,although poor Glasgowcoma scale(GCS)scores,intubation,dysphagia and invasive procedures are associated.Further,autonomic shift,as detected by decreased baroreflex sensitivity,was associated with increased susceptibility to infection in ICH patients.Infection,in turn,contributes to negative outcomes of ICH patients,including death.Additionally,lymphopenia after ICHcan signal the likelihood of a poor outcome.In an animal model,ICH appeared to impair peripheral cellular immunity.Despite thebackground above,how ICH impacts the immune system,the underlying mechanisms and its association with patients' outcomes remains obscure.Therefore,we sought to investigate alterationsin the immune system at the organ and cellular levels of subjects with ICH and to determine the neurogenic pathways causing such alterations.Relevance of the immune response with spontaneous infection and outcomes of ICH patients were analyzed.Materials and methods:1.ICH participants:ICH patients were recruited for this study: men and non-pregnant women ? 18 years of age with basal ganglia hemorrhage,ICH of 5-40ml(confirmed by CT),symptom onset < 24 hours prior to hospital admission,a GCS score of ? 6.Exclusion criteria included patients with a GCS of 3 to 5,planned surgical evacuation,infection within 2 weeks of admission after onset.Also excluded were patients with hematoma expansion,secondary ICH,concomitant use of antineoplastic,immunosuppressive or immune-modulating therapies.Healthy controls were matched by age,gender and body mass index.2.spleen and brain MRI :All eligible subjects had a spleen and brain MRI at days 3 and 14 after ICH diagnosis.Brain MRI performed T2/T1/FLAIR/DWI,identifide volume of ICH and PHE.Each spleen's volume was measured on a T2 image of the abdomen.Intravoxel incoherent motion(IVIM)diffusion-weighted(DW)imaging19 was performed to calculate the static tissue molecular diffusion(D,true diffusion coefficient,which reflected cell activity)and perfusion-related pseudodiffusion(D*,pseudodiffusion coefficient,reflecting the velocity of capillary blood).PHE was quantified by FLAIR images of the brain.3.Quantification of lymphocytes in the circulation:Lymphocyte subset analyses were performed on fresh whole-blood samples from all human participants.Isolated mononuclear cells were immunostained withfluorochrome-conjugated antibodies.The following antibodies to human antigens were used: CD3-Per CP,CD4-FITC,CD8-PE,CD19-PE,CD56-APC,CD11b-Per CP,CD11c-APC.Data were acquired using a FACSCalibur and analyzed with Flow Jo software.4.neurotransmitters and stress hormonesin the circulation:Plasma levels ofthe neurotransmitters acetylcholine and norepinephrine and the glucocorticoid hormone cortisol were measured by enzyme-linked immunosorbent assay(ELISA)according to the manufacturer's instructions.Optical densities were measured at 450 nm and 570 nm.5.Induction of the ICH modelgous blood in the right basal ganglia site described previously:Adult male C57BL/6 mice(10-12 weeks old)were used for the animal experiments.For the collagenase model,ICH was induced by intracerebral injection of collagenaseat the site of right basal ganglia;to induce ICH of varied volumes in mice,graduated doses of collagenase were used.For the autologousblood injection model,mice were injected with 30?l autolo6.Test content of the ICH model:Spleens of ICH mice were weighed at indicated time points immediately after sacrifice.For pathological staining,procedures were completed as described.Briefly,mice were sacrificed at day 3 after ICH induction;spleens were quickly removed and fixed in 4% paraformaldehyde overnight followed by dehydration with 30% sucrose until the tissue precipitated to the bottom.Incubation in Optimal Cutting Temperature(O.C.T.)compound followed.Then,8?m-thick cross-sections were cut on a cryostat and mounted on poly-L-lysine-coated slides.Each tissue section was stained with hematoxylin-eosin(HE)to measure the white and red pulp areas of the spleen.Pictures were acquired with a fluorescence microscope and analyzed using Image Pro Plus.7.Neuropathway blocking experiments in mice:For neuropathway blocking experiments in mice,the following antagonist regimens were used: RU486 was dissolved in ethanol/sesame oil solution(1:10,6 mg/ml)and injected i.p.at 30 mg/kg body weight immediately after ICH.Propranolol was dissolved in saline at 6 mg/ml and injected i.p at 60 mg/kg immediately after model induction.Dihydro-?-erythroidine(DH?E)was dissolved in saline at 0.1 mg/ml and1mg/kg was injected i.p.immediately after ICH induction.8.Statistical analysis:Statistical analysis was performed using SPSS version 17.0 software for Windows.The results are expressed as mean ± SD for continuous variables and as a probability for categorical variables.Between two groups,non-parametric data were compared byusing the Mann-Whitney test,and parametric data were compared with Student's t-test.Additionally,a one-way analysis of variance(ANOVA)was performed for comparing parametric data among more than three groups with post-hoc tests;two-way ANOVA was used to compare repeated measurement-to-data between two groups.The Chi-square test was used to analyze differences in categorical variables.Spearman correlation analysis was conducted to assess factors that may contribute to the occurrence of spleen and lymphocyte alteration.A value of P < 0.05 was considered significant.Results:1.Characteristics of ICH patients upon admission:A total of 35 patients with ICH were recruited into the present study between August 2015 and May 2016.There were no fatalities,no loss at follow-up and no drop outs.The mean time from onset toenrollment was 11.7 ± 8.5 hours.The mean age of patients enrolled was 62.1 ± 11.8 years,and the male:female ratio was 27:8.The 20 healthy controlshad a mean age of 59.1 ± 9.8 years and male: female ratio of 15:5.Demographic,clinical and radiological characteristics,age,genderand body mass index(BMI)in the patients with ICH and healthy controls were similar.2.Dramatic spleen shrinkageafter ICH,capillary blood perfusion ofthe spleen had increased,although no apparent alteration of splenocyte activity:Spleen volumes of ICH patients at day 3 after ictus were evaluated.At that time,their spleens were smaller thanat day 14;in fact at day 14,their spleen volumes resembled the sizein healthy controls.The average spleen shrinkage volume,then,was37cm3,which translated into a 17.2% decrease in average spleen size at day 3 after ICH.IVIM-DWI models the diffusion-attenuated MRI signal was a sum of static tissue molecular diffusion and perfusion-related pseudodiffusion.D* and D values for the spleen were not significantly different between healthy controls and the ICH patients at day 14 after disease onset.However,the patients' D* value was significantly higher at day 3 than day 14in(p<0.001),whereas the D value was stable.3.Spleen shrinkage can be predicted by hematoma size at admission and relates to PHE and ICH outcomes:patients were arbitrarily divided totwo groups: severe spleen responsewith spleenshrinkage> 37cm3(n=16)or mild spleen response with spleen shrinkage < 37cm3(n=19).That is,the mean hemorrhage volume at admission for patients with severe spleen responses was larger than for patients with mild spleen responses(12.9 ± 10.5 vs 8.2 ± 6.5 ml,p < 0.05).Moreover,we found hemorrhage volume at admission(<24 hours)was associated with spleen shrinkage at day 3 after ICH(r = 0.48,P < 0.01).the PHE in patients with mild spleen responses expanded rapidly in the first 3 days post ICH,and that trend continued up to14 days.In contrast,patients with severe spleen responses had milder and slower expansion of edema volume in the 3-day post-ICH period and that level remained essentially the same during the following11 days.Quantitatively,relative PHE(r PHE)was smaller in patients with a severe spleen response than in those with a mild spleen response both at day 3 and day 14 after ICH(2.8 ± 1.5 vs 4.5 ± 1.5,P < 0.001;3.2 ± 1.5 vs 5.8 ± 2.5,P < 0.01).Although the NIHSS score from admission to 90 days did not differ significantly between patients withmild and severe spleen responses,the decreasein score from admission to 90 days was larger in patients whose spleensshrank> 37cm3(P< 0.05).Therefore,patients with severe spleen shrinkage might have relatively better recoveries from their neurological deficit.However,the modified Rankin Scale(m RS)0–1 at 90 days between the two groups did not reach the status of significant difference.4.Deficiencies of T and naturalkiller(NK)cells after ICH were associated with increased susceptibility to infection and poor outcomes:Numbers of lymphocytes and of CD4+T,NK,B,and CD11C+ cells decreased after the onset of ICH.Except for one subset,all these cell counts reached a nadir at day 3 and persisted at that low level untilday 14.Although the CD8+T cellsubsetsimilarly displayed a negative trend after ICH,the difference was notstatistically significant.In addition,CD4+ T and B cell countsof 3 days were negatively associated with hematoma volume at admission,but total lymphocyte and other cell subunits didnot show such an association.Neither the full lymphocyte spectrum nor the subunits differed significantly in cell counts of patients with spleen shrinkage> 37cm3 and patients with spleen shrinkage< 37cm3.Of these 35 subjects,12(34%)suffered infection during hospitalization.At admission,the hematoma size of patients who became infected seemed to be larger than in patients without infection;however,such differences didnot reach statistical significance(13.5 ± 9.5 vs 8.7 ± 6.7 cm3,P>0.05).70% of patients without infectionshad better outcomes versus only 8% of infected patients with better outcomes(P <0.0001).Additionally,CD4+T,CD8+T,and NK cell counts were significantly lower in patients with infection than withoutfrom days 1 to 14.5.A synergy of sympathetic and hypothalamus-pituitary-adrenal gland(HPA)axis activation mediated the spleen response after experimental ICH:We then used an experimental model of ICH in mice to test the foregoing clinical outcomes of patients.To ensure confirmation,two kinds of models were used.Their spleen weights significantly decreased.As a result,the larger the dose of collagenase applied,the larger was the hematoma induced,and the more obvious was spleen shrinkage of these mice.HE staining of spleens from ICH mice showed that the red pulpremained segregated from the white pulp in sham-operated mice,and the red pulp did not brim over its separate space.However,in the ICH mice,blood brimmed sufficiently that a large number of erythrocytes were visible inside the vessels.The white pulp areas were rarely seen and not clear in ICH mice compared to those in the sham-operated mice(p<0.05).We used the ?-adrenergic receptor blocker,propranolol,for adrenergic blockade,RU486 for glucocorticoid receptor blockade,and ?4?2 cholinergic blocker DH?E for cholinergic blockade.Our results showed that,DH?E failed to block the splenicresponse,butpropranolol and RU486 partiallyinhibited the spleen shrinkage of ICH mice.In addition,the neurotransmittersdetectedhere showed that the acetylcholine levelsin plasma were lower in ICH patientsthan in healthy controls and remained stable from days 3 to 14,while the concentrations of norepinephrine and cortisol were higher in ICH patient serum at days 3 and 14.Together,these results suggest a synergistic pattern of sympathetic innervation and the HPA axis in the ICH-induced immune response.Conclusion: 1.Dramatic spleen shrinkageafter ICH,capillary blood perfusion ofthe spleen had increased,although no apparent alteration of splenocyte activity.2.Spleen shrinkage can be predicted by hematoma size at admission and relates to PHE and ICH outcomes.3.patients with specific deficiencies in cellular immunity had increased susceptibility to infection.4.A synergy of sympathetic and hypothalamus-pituitary-adrenal gland(HPA)axis activation mediated the spleen response after experimental ICH.5.Spleen shrinkage and lymphopenia,reflecting the impact of ICH on the immune system at the organ and cellular levels,derived from the coordinated actions of sympathetic innervation and HPA axis. |
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