The Mechanism Of Early Perihematoma Edema Formation After Intracerebral Hemorrhage

Background and purpose:Intracerebral hematoma (ICH) has a higher mortality rate and produces more extensive neurological deficit than any other type of stroke. Perihematomal edema after ICH is the main reason of that, it may induce degeneration and necrosis in neurons and axons. But its pathophysiological mechanism (especial in the early stage ) is very complicated and poorly understood. It has influenced the treatment plan seriously. So it has much significance of theory and clinical value. At present,Much hypothesis about the mechanism of perihematoma edema formation remains still a matter of debate: one is an ischemic mechanism for edema formation, the authors thought that hyperacute perihematoma edema was cytotoxic edema. However, recent evidence from experimental and clinical studies did not support this view, they thought that the early perihematoma edema was vasogenic du to BBB broken. And some scholar thought that the early perihematoma edema is caused by plasma derived and clot retraction. The focus of the hypothesis is what causes the early perihematomal edema? Decreasing of rCMRglc or increasing of blood capillary permeability. This study studed the changesed the function, morphous and metabolisim of perihematomal edematous tissues to reveal the mechanism for perihematomal edema formation with MR DWI, PET/CT,biologic chemistry and histopathology in ICH model of cats.Part Ⅰ : Establishment and evaluation of feline model of intracerebralhemorrhage under the MRI location Objective: To establish animal models of intracerabral hemorrhage, whichcan fit for clinical MRI and PET imaging with small animal for large sample studying.Methods: 18 elder cats(male, weighted 3.0 ± 0.2kg ,supplied by experimental animal center of Shandong university) were used in the experiment, Animals were randomly assigned into three groups (each group 6 cats )and anesthetized by an intraperitoneal injection of thiopental (40 mg/kg). Each cat was placed in the prone position with its head fixed in an operating frame and underwent MR scanning on three dimensions to measure the distance between ROI posterior to anterior fontanelle(A),distance between ROI to the left of the sagittal suture(L)and the distance between ROI to deep to external lamina(H) of the head of caudate nucleus, internal capsule and thalamus. Each animal underwent a stereotactic injection of 1 ml of fresh nonheparinized arterial blood from the femoral artery into the Rol according to the MR measurements. At 2 hour after operation, the ICH models underwent MR scanning and PET/CT scanning to evaluate the normal brain structure, the MR features of the hematoma and the 18F-FDG intensity in brain.Results:1. Details of the gross anatomy of the cat brain were distinguished best in the FDG images and MR images. CMRgluc values measured in the cortex, white matter and basal ganglia were within the range of normal autoradiographic results.2. The The scores for measurements (mean ±SD, mm ) of the head of caudate nucleus, internal capsule and thalamus are respectively.- A: 5.8±0.4, 11.1±1.2, 11.9±1.3; L: 5.5±0.5, 7.1±0.6, 7.2±0.7; H: 16.6±1.4,19.6±1.8,21.5±2.1;3. On MR images, the typical hematoma with clear boundary in region of interest (Rol) can be found in all ICH models, hematoma volumes is about 0.42 ± 0.13ml. On PET images, Immediately after ICH, there was widespread decrease in metabolism of glucose.Conclusions:1. Clinical MRI and and 18F-FDG PET scanner can not only show details of the gross anatomy of the cat brain clearly ,but also reflect the typical MRIfeatures of hematomas and changes of CMRgluc in the brain after ICH, which demonstrates that a cat is ideal animal which could be fit for the clinical MRI and PET for experimental neurological imaging studying.2. The hematomas in ICH models localized under MRI were stable and exact, which means that localization under MRI is an effective method for ICH models establishment.Partll: The MRI study on the mechanism of early perihematomal edema formation after intrcerebral hemorrhageObjective: To investigate the mechanism of perihematomal edema formation and developing from super acute stage to early subacute stage of ICH with the technique of diffusion weighted MR imaging (DWI) ; To assess the role of the DWI technique for hematoma diagnosis in hyperacute stage to early subacute stage of ICH and to answer whether existing a penumbra around the clot.Methods: 54 cats were randomly assigned into nine groups: one sham operation group and eight operation groups: 2h,6h,12h, 24h, 48h, 72h, 120h and 148h groups. Each group was cinsisted of 6 cats. And the cats in 2h group underwent 3 time points MR scanning: 20min, lh and 2h after operation ; the cats in 6h group underwent 2 time points MR scanning: 3h and 6h after operation. ICH models were established as "part 1" , the sham operation group was performed the same operation as operation groups except infusing blood, hyperacute stage included 20min, lh, 2h, 3h and 6h time points, acute stage included 12h, 24h, 48h and 72h time points, and early subacute stage included 120h and 148h time points. All the animals underwent MRI scanning.MRI was performed with the following sequences : Tl weighted imaging (TlWI) , T2 weighted imaging (T2WI) , fluid attenuated inversion recovery (FLAIR) , diffusion weighted imaging (DWI) and GD-DTPA contrast enhanced T1WI ( CE-TiWI) . The relative signal intensity, apparent diffusion coefficient(ADC) of the clot and Perihematomal edema tissue were measured. Edema and hematomal volumes were measured on T2-weighted images (ABC 112).The reviewers observed the following parameters through MR imaging as: the changes of volume and the relative signal intensity, apparent diffusion coefficient (ADC) of the clot and Perihematomal edema tissue were observed; the volumes of edema and hematoma were measured on T2-weighted images (ABC 1/2). Results:1. The changes of MRI signal intensity and hematomal volume in different ICH stage.Hematomas showed markedly central hyperintensity accompanied by a hypointense rim in hyperacute stage on T2-weighted images, hyperintensity was also can be found on FLAIR as well as DWI(bO), it decreased with time and reached the lowest point at 24-48h after ICH, then increased gradually. Hematomas were always hypointense at DWI(bl000) in our study, but its ADC value was very higher (rADC=98.8) at 20min after ICH, then it reduced sharply and keep lower level than that of normal contralateral white matter. On Tl-weighted images, the volume decreased by 22%, intensity of the hematomal core increased byl9%, the hypointense outer lice around the core enlarged from 20min to 6h, which imply a clot retraction.2. The changes of MRI signal intensity and volume in different stage of perilesional edemaPerilesional edema seen on T2-weighted images to surround the hematomas showed a 1-mm-wide hyperintense area around the clot at 20min after ICH, its volume increased 11.8% in hyperacute stage, increased by 229.4% at 24h, and to its peak (increased by 273.5%) at 72h after ICH, then decreased. Its T2WI intensity changed liked this, and there was a significant association (P<0.05, r=0.97)of intensity with volume from onset not with ICH size. The hyperintense area of perilesional edema seen on T2-weighted images was also seen on FLAIR and DWI images, it has a higher ADC than that of normal contralateral white matter, and there was a significant association (P<0.05, r=0.95) of ADC value with edema volume too. Meanwhile The hyperintense area was thinner onDWI(blOOO) images than that on T2-weighted images, and not consistent with edema volume. The GD-DTPA contrast enhanced T1WI showed high intensity around clot appeared at 12 hour in acute stage, it was not obviously in the early subacute stage. Only one little region of high DWI with low ADC below the hematoma can be seen in a ICH model. Conclusions:1. Perihemotomal edema of the hyperacute stage was neither traditional vasogenic edema nor cytotoxic edema, it was due to clot retraction ant serum protein (derived from the clot) accumulation in brain parenchyma. Acute and subacute perihemotomal edema was due to increased blood-brain barrier permeability.2. Hyperacute hematomas have characteristic MRI features on T2WI, DWI and its ADC, which shows that modern MRI alone can diagnose ICH and demonstrate the underlying pathophysiology independently.Pai-tHI: The 18F-FGD PET study on the changes of early glucose metabolism and its influence on perihematomal edema formation after intracerebralhemorrhageObjective: To study the changes of glucose metabolism and its influences on perihematomal edema, from hyperacute stage to early subacute stage With the technique of 18F-FGDPETMethods: The 48 cats and its groups of sham operation group, 2h, 6h,12h, 24h, 48h? 72h and 120h operation groups in "Part 2" were used in this experiment The grouping of animals were according to "Part 2": one, each group has 6 cats. ICH model made as "part 1" , sham operation group was performed the same operation as model group except infusing blood. All animals underwent 18F-FDG PET/CT scanning after MRI scanning in Part 2. The location of ROI on PET images was confirmed by the relative MR images Results:1. 18F-FDG intensity in perihematomal edema tissues was markedly low at2h after ICH on PET images, and it decreased to the lowest level at 6h, keep lower level from 24h to 48h, it began to restore at 72h, at 120h the 18F-FDG intensity was close to control group.2. The SUV in perihematomal edema changes was consistent with the 18F-FDG intensity. At 2h, it was 0.97+0.07 (vs contralateral 1.33 + 0.15, P<0.05), at 6h it was 0.93±0.15(vs contralateral, PO.05), at 72h, it increased to 1.83 + 0.155(vs 1.86+0.14, P<0.05). Low 18F-FDG intensity and SUV were also seen in bilateral frontal lobe and other areas of brain on FGD PET images.Conclusions:1. In Hyperacute ICH(2h,6h), cerebral metabolitic rate of glucose was at an obvious low level, it was a primary metabolitic depression..2. The changes in perihemotomal edema and metabolism can be followed over time. There was a significant association of changing of metabolitic rate and edema formation with time from onset, and the former was 24~48h ahead of the latter. It demonstrate that perihemorragic metabolism change was unrelated to hyperacute perihemotomal edema, it was related to acute and subacute vasogenic edema formation. Abnormal glucose metabolism maybe destroy capillaries integrity and increase blood-brain Barrie permeability.PartIV The biochemical study on early changes of metabolism in perihametomal edematous region after intrcerebral hemorrhageObjective: To observe the change of cerebral water content, lactate content and LDH activation and their influence on perihametomal edema formation in ICH model in feline.Methods: The grouping of animals were according to "Part 2". Animals were killed corresponding to test time by overdose anesthesia, break head and take brain immediately, take lg cerebral tissue surrounding the hamotoma for evaluating its water content by dry-wet weight method , the formula was.- water content=(wet weight - dry weight)/wet weight. Take Take out another lg cerebral tissue for lactate and LDH testing.Results:1. The changes of cerebral water content Cerebral water content on perihematomal edema is compared with those in the one sham operation group.The cerebral water content began to increase at 2h (P<0.05), to its peak at 48~72h and restore at 5d.2. The changes of cerebral lactate and LDH. The cerebral lactate in perihematomal region began to increase at 2h(P<0.05), to its peak at 24h and still keep high level at 72h, which is markedly different from those of control group. The LDH began to increase at 6h(/><0.05), to its peak at 72h and restore at 120h.Conclusions:1. Cerebral lactate increasing was associated with the dysbolism of glucose, not with anaerobic glycolysis, and it was perhaps due to glial cell proliferation. Cerebral lactate increasing destroyed the BBB in acute stages and caused vasogenic edema.2. The changes of LDH was later than that of lactate.The LDH began to increase at 6h(p<0.05), to its peak at 72h then went back to normal, which means the increasing of LDH was activated by lactate.Part V The histopathologic study on early perihematomal edema formation after intracerebral hemorrhageObjective: The purpose of this study is to observe the histopathologic changes of the cerebral tissue in perihematomal edema in hyperacute stage and acute stage and to investigate the mechanism of perihematomal edema formation after ICH.Methods: 36 ICH cats of sham operation group and 2h, 12h, 24h, 48h and 5d operation groups were used in this experiment. The taking of samples was as "Part 4". Take a lcm3 cerebral tissue to stained in 10% methanal, routine embedding, section, dyed by HE and observed under microscope and photographed. Take lmm 3 cerebral tissue stained in 2% glutaral, desiccation by 70%~100%alcohol and acetone, Epon-812 routine embedding, stabilization by 1% osmium acid, and observed by 1200EX transmission electron microscope andphotographed. Results:1. light microscopic observationHyperacute stage: Blood vessel integrity, neuroglial cells proliferation, mesenchyme was relaxed, the nucleus of neurons blured were be seen, no exudates seen around the vessels. Acute stage: 12h after ICH: mesenchyme was swelled obviously, and more exudates could be seen around the vessels. 48h-72h after ICH: blood vessels collapsed. Early subacute stage: organization of hematoma, Vascular proliferation and gitter cell could be seen.2. eletric microscopic observationSham operation group: The nucleus of neurons are big and circular, nucleolus was obvious, chromatiri of nucleus was homogenous, organelle was plentiful in cytoplasm, the nucleus of neuroglial cells was circular, no abnormality shown in surroundings, and the structure was normal. The structure of vessels and endothelial cells were normal, and no exudates seen around the vessels. Hyperacute period group(2h): The basement membrane integrated, the Peripheral region of microvessel presented slight edema; the cell body of neuron presented mild swelling nuclear membrane integrated; nucleolus distinct chromatin relaxed; cytoplasm relaxed and crevice was presented. Acute period group(12h/s-'48h): The basement membrane broke, the Peripheral region of microvessel presented obvious edema; nucleus of neuron contract; The chromatin of nucleus condensed block shape; cytoplastic relaxed; organell swelled; mitochondrion swelled and vacuolatedendoplasmic reticulum swelle, neuroglial cells process and swelled; and endothelial cells were proximately round.Conclusions: In hyperacute ICH, the BBB was integrity, the perihematomal edema wasinterstitial edema; In acute ICH, the BBB was broken, the perihematomal edema was vasogenic edema.Conclusions and Significances1 .The study described the changing course of the perihenatomal edema on ADC, cerebral glucose metabolism, histomorphology detailedly and systematically from 20mins to 7 days after ICH with DWI, PET/CT, biochemistry and histopathology. For the first time, the study confirmed the formation mechanism of hyperacute perihematomal edema and the important role of abnormal cerebral glucose metabolism in it. The results will play an important role in clinical treatment strategies and ICH experimental study.1.1 Hyperacute perihematomal edema was due to clot retraction and serum protein derived from clot accumulation in brain parenchyma.Evidence: ?The volume of clot becoming smaller and ADC decreasing abruptly reflected the course of serum protein deriving from clot; The increasing of ADC in perihematomal edema mean that the perihematomal edema was interstitial, not cytotoxic. (2) Traditional vasogenic edema was ruled out by none Tl WI enhencement and the Integrity of BBB in perihematomal edema.1.2 Acute perihematomal edema was vasogenic edema due to BBB broken. Evidences: ?The positive correlation between ADC and T2WI intensitydemonstrated that the edematous water exited out of the cells. ? The integrity of BBB was corformed by positive CE-T1 WI and electron microscope .1.3 Subacute perihematomal edema was vasogenic edema.Evidences: ?The ADC value, T2WI intensity, edematous volume and water content increased to its peak at 5th day and decrease at 7th day. (2) None T1WI enhencement and organization of hematoma demonstrated that the damaged tissue began to restore.1.4 Abnormal cerebral glucose metabolism plaied an important role in perihematomal edema formation, it may influence the Integrity of BBB and it was an important complement to edema formation theory, although it need further clinical and experimental test.Evidences: ? Cerebral glucose metabolism decreasing, lactate increasing at 2h after ICH, none anoxic evidence or none BBB broken demonstrated that tissue utilize disordered. (2) Cerebral glucose metabolism supressing and lactate increasing occurred 24h ahead of BBB broken, Cerebral glucose metabolism increasing and lactate decreasing also it dependent occurred 24h ahead of edema extinct.2. The study built up a new method foe establishment of the ICH model and broken the limit of traditional method. The method has importanr significance to develop neuroimaging research.2.1 The method of the establishment of the ICH model in feline localized under MRI was direct, visual, exact and feasible, firstly put into practiced , it will rich the ways of establishment ICH models.2.2 .The sectional data of ICH model in feline obtained in this study was a necessary complement to the traditional atlas of feline brain, its reliability has been confirmed by this study.2.3 For the first time , we do a research on the same model with DWI, PET/CT, biochemistry and histopathology. The method resolved the deviation of results caused by single means.2.4 Our study demonstrated that the brains of cats can meet clinical MRI and PET scanning well, the results will improve experimental research for cerebral vascular diseases with modern clinical imaging equipments.3. The conclusion that "modern MRI can diagnose and demonstrate the underlying pathology of the hyperacute ICH independently" is a breakthrough compared with traditional diagnostic model of cerebral vascular diseases. No matter ICH or ischemia, MRI can make a diagnosis almost at the same when stroke onset, it was significant for exact ICH treatment in phase .