Pathology Study On Piping Phenomenon After Cerebral Hemorrhage

Cerebral hemorrhage is a frequent and common disease, with high mortality and disability. After cerebral hemorrhage, a series of pathological changes will occur in adjacent cerebral tissues of the hematoma, such as edema and ischemia of cerebral tissues, etc. In recent years, many studies on pathological mechanism of cerebral hemorrhage have been conducted consecutively, but with few breakthroughs. It has already been found that punctate hemorrhage lesion was observed in tissues close to the hematoma, these hemorrhagic lesions usually surrounded blood vessels in ring shapes, so it was also named "ring hemorrhage". It is still not clear about its pathological mechanism, and commonly it is believed that, disorder of venous return is induced by depression of hematoma, and digestive infarct occurs in cerebral tissues, and blood would overflow from the small vessels. It has been observed in our preliminary studies that, a unique pathological phenomenon-"piping" exists in the adjacent tissues of hemorrhagic area, which is caused by distant extension of liquid blood along the Virchow-Robin spaces (VRS) and nerve fibers under pressure gap."Ring hemorrhage" is actually induced by external distension of hematoma along VRS. "Piping phenomenon"may be correlated with distant effect of hematoma, lymphatic cerebral edema, secondary subarachnoid hemorrhage and secondary cerebro-ventricular haemorrhage, etc. and further confirmative studies are necessary.Ⅰ. Experimental study on cerebral hemorrhage rat model constructed by injecting autologous blood into caudate nucleus with the method of double-injection through preset canulaObjectiveCanula was preset to inject autogeneic arterial blood for two times to prepare cerebral hemorrhage rat model, with non-preset canula cerebral hemorrhage rat model as the control, thus to investigate the feasibility of preset canula technique to inhibit needle contra-flow, and improve the stability of cerebral hemorrhage in animal models.Materials and methodsPaired design was adopted to take 12 pairs of health male SD rats(250±10g), the pairs had similar body weights, and were from the same brood, same feeding conditions were adopted, and the rats were randomized into preset group and non-preset group, with 12 rats in each group. Rats of preset canula group were loaded with catheter under stereotaxic apparatus after 10% chloral hydrate intraperitoneal anesthesia,50μL blood was collected from femoral artery 24h after the operation and injected gradually into caudate nucleus, and then the canula was closed. Rats of non-preset group were loaded with central canula in left caudate nucleus under stereotaxic apparatus, and then 50μL blood was instantly and gradually injected. All rats were executed by injecting 10% chloral hydrate 24h after injection of autogeneic arterial blood, the tissues were fixed by using 4% paraformaldehyde infusion, then integrate cerebral tissues were collected and immersed in 4% paraformaldehyde infusion for 48 h to prepare series sections of cerebral tissues. The slice thickness was 2 mm. Images were taken after the slices were observed with naked eyes. Then frozen section was conducted and images were taken under microscope after HE stained.ResultsHematoma can be observed in left caudate nucleus of rats in two groups, the average hematoma volume (25.1±0.5)μL of preset group is significantly higher than that of control group (24.2±0.7)μL. The dispersion coefficient of hematoma volume is 2.0% in preset group, obviously lower than 2.9% in control group. The hematoma is round and regular in preset group, with apparent occupation effect.Ⅱ. Pathology study on "piping phenomenon" of formed elements in hematoma after cerebral hemorrhageObjectiveTo investigate the distribution of "piping phenomenon" of formed elements in hematoma at different times after cerebral hemorrhage with isothiocyanate (FITC) fluorescent probe labeled red blood cells. Materials and methodsPreset canula was adopted to inject autologous arterial blood and prepare rat model with hemorrhage of caudate nucleus. A total of 40 male rats weighing 250±10 g were selected and randomized into 6 test groups,2 sham operation groups (1 h group,3 d group), with 5 rats in each group. The six test groups included 1 h group,6 h group,24 h group,3 d group,7 d group and 14 d group.Preparation of fluorescein isothiocyanate (FITC) probe labeled autogenous arterial blood:FITC labeled autologous erythrocyte (FITC-RBC) was adopted and with autologous serum re-suspended to the original volume, and take 50μL to prepare animal model with cerebral hemorrhage.Preparation of cerebral hemorrhage animal model by using FITC labeled arterial blood:Adopt rat model with cerebral hemorrhage prepared by using double injection of autologous arterial blood with preset catheter. The catheter was preset into the center of caudate nucleus under a stereotaxic apparatus,24 h later, inject 50μL FITC labeled autogeneic arterial blood through the catheter to construct a cerebral hemorrhage model.Sham group only puncture needle to center of the caudate nucleus, do not inject blood.Preparation of tissue sections and observation of sections florescence and HE staining:Rat models of different tests groups were anesthetized and executed at corresponding time points, then the blood vessels were infused and tissues were fixed. Rats in sham operation groups were anesthetized and executed 1 h and 3 d after operation, then the blood vessels were infused and tissues were fixed. Brain tissues, as well as bilateral deep cervical lymph node were separated.A blue light of 460-490 nm was used to excite, and routine HE staining was conducted after fluorescence photographing, and then observed and photographed under light microscope. The florescent images and HE images of the same site were input into Photoshop CS4, and the images were overlain and analyzed after artificially adjusted.ResultsFITC-RBC was mainly distributed in the tissues surrounding hematoma extensively along blood gaps and nervous fibers, and usually extended to callositas of the same lateral. It was also distributed in basal ganglia of the same lateral and VRS of cerebral cortex, FITC-RBC was also distributed in spaces adjacent to blood vessels in brain stem and contra-lateral hemisphere. Liberated FITC could be observed in tissues surrounding hematoma after FITC-RBC schizolysis, and more could be observed if the hematoma was formed longer. Liberated FITC was distributed more extensively than FITC-RBC, and inVRS and areas under meninx vasculosa in brain stem. When the hematoma has formed for 1 h, phagocyte containing yellow brown granules appeared in VRS in different cerebral areas, and the lesion was most significant. The distributed area of hemosiderin became more as time passed, and it covered the perivascular spaces of all areas in the brain 14 d later.When hematoma has formed for 1 h, FITC-RBC and liberated FITC could be observed in lymphonodi cervicales profundi of both sides similarly,and mainly distributed in lymph sinuses. No FITC-RBC and liberated FITC had been observed in germinal center of lymph nodes. As time passed, FITC-RBC decreased gradually, but liberated FITC and yellow florescent granules increased significantly.Sham group rat brain tissues and bilateral deep cervical lymph nodes showed no granular distribution of fluorescent substances. Ⅲ. Pathology study on "piping phenomenon" of formless elements in hematoma after cerebral hemorrhageObjectiveTo investigate the distribution of "piping phenomenon"of formless elements in hematoma at different times after cerebral hemorrhage with Tetramethyl Jiluodanming (TAMRA) fluorescent probe labeled bovine serum albumin (TAMRA-BSA)Materials and methodsPreset canula was adopted to inject autogeneic arterial blood and prepare rat model with hemorrhage of caudate nucleus. A total of 40 male rats weighing 250±10 g were selected and randomized into 6 test groups,2 sham operation groups(1 h group,3 d group), with 5 rats in each group. The six test groups included 1 h group,6 h group,24 h group,3 d group,7 d group and 14 d group.Preparation of Tetramethylrhodamine (TAMRA) florescent probe labeled autologous arterial blood:TAMRA florescent probe labeled bovine serum albumin (TAMRA-BSA) was adopted and mixed into autologous arterial blood of rats, to adjust the final concentration of TAMRA-BSA to 40mg/ml, then 50μL TAMRA labeled autologous arterial blood was collected to prepare animal model with cerebral hemorrhage.Preparation of cerebral hemorrhage animal model by using florescent probe labeled arterial blood:Adopt rat model with cerebral hemorrhage prepared by using double injection of autogeneic arterial blood with preset catheter.The catheter was preset into the center of caudate nucleus under a stereotaxic apparatus.After 24 h, inject 50μL TAMRA-BSA labeled autogeneic arterial blood through the catheter to construct a cerebral hemorrhage model by using TAMRA florescent probe labeled arterial blood.TAMRA-BSA labeled blood was not injected to rats of sham operation control group.The needles were only inserted into the caudate nucleus of rats in sham operation control group, and arterial blood was not injected.Preparation of tissue sections and observation of sections florescence and HE staining:Rat models of different test groups were intra-abdominally anesthetized, fixed by using 4% paraform and executed at corresponding time points.Rats in sham operation groups were executed 1 h and 3 d after operation.Cerebral tissues and bilateral lymphonodi cervicales profundi were stripped out, freeze sectioned, and then observed under fluorescence microscope.Green exciting light of 510-550 nm was used, and routine HE staining was conducted after fluorescence photographing. The florescent images and HE images of the same site were input into Photoshop CS4, and the images were overlain and analyzed after artificially adjusted.ResultsTAMRA-BSA extensively distributed in cerebral tissues in VRS of all areas in the brain. A large number of TAMRA-BSA granules existed in hematoma and adjacent tissues, and TAMRA-BSA granules also distributed in the VRS of basal ganglia and cortex of the same time, and also in the VRS in brain stem and contra-lateral hemisphere.TAMRA-BSA granules could also distribute along nerve fibers in near distance, only in the adjacent tissues, and the usually seen sites included nervous fibers close to the hematoma and callositas.TAMRA-BSA granules distributed similarly in different cerebral areas and at different times after hematoma formed. After 1 h, TAMRA-BSA granules could be observed in inter-vascular spaces and distributed extensively in spaces between nerve fibers on the same lateral of lesion, and distribution of TAMRA-BSA granules was similar at other periods.When hematoma has formed for 1h, TAMRA-BSA florescent granules could be observed in lymphonodi cervicales profundi of both sides equally.The granules distributed mainly in lymph sinuses, and TAMRA-BSA florescent granules could also be observed in the germinal center of lymph nodes.The distribution of TAMRA-BSA florescent granules in lymph nodes were basically the same at different time points.When hematoma has formed for 6 hours, the lesion began to have yellow brown florescent granules-phagocytic cell, as time passed, yellow brown florescent granules-phagocytic cell could also be observed in VRS of bilateral cerebral hemispheres and brainstem, mainly around hematoma. As time went by, brown color was gradually deepened.No distribution of granular fluorescent substances in the brain tissues of sham group rats. Bilateral deep cervical lymph nodes also showed no physical distribution of particle fluorescence.Sham group rat brain tissues and bilateral deep cervical lymph nodes did not showe granular distribution of fluorescent substancesIV. Pathology study on relationship between "piping phenomenon" and the lymphatic encephalopathyObjectiveTo investigate the relationship between the "piping phenomenon" and " Lymphostatic cerebral edema" with FITC labeled red blood cell, TAMRA labeled BSA on the bases of preliminary study on "cerebral edema Lymphostatic" after cerebral hemorrhage.Materials and methods:Preset canula was adopted to inject autogeneic arterial blood and prepare rat model with hemorrhage of caudate nucleus. A total of 70 male rats weighing 250±10 g were selected and randomized into 6 TAMRA test groups,6 FITC test groups,2 sham operation groups (1 h group,3 d group), with 5 rats in each group. Each test group included 1 h group,6 h group,24 h group,3 d group,7 d group and 14 d group.Preparation of fluorescein isothiocyanate (FITC) probe labeled antogenous arterial blood: EITC labeled autologous erythrocte (FITC-RBC) was adopted and re-suspended to the original volume with autologous serum, and take 50μL to prepare animal model with cerebral hemorrhage.Preparation of Tetramethylrhodamine (TAMRA) florescent probe labeled autologous arterial blood:TAMRA florescent probe labeled bovine serum albumin (TAMRA-BSA) was adopted and mixed into autologous arterial blood of rats, to adjust the final concentration of TAMRA-BSA to 40 mg/ml, then 50μL TAMRA labeled autologous arterial blood was collected to prepare animal model with cerebral hemorrhage.Preparation of cerebral hemorrhage animal model:Adopt rat model with cerebral hemorrhage prepared by using double injection of autologous arterial blood with preset catheter. The catheter was preset into the center of caudate nucleus under a stereotaxic apparatus,24h later, inject 50μL FITC labeled autogeneic arterial blood and 50μL TAMRA-BSA labeled autogeneic arterial blood through the catheter to construct a cerebral hemorrhage model.The rats in Sham control groups were not injected with FITC labeled autologous blood and TAMRA-BSA labeled autogeneic arterial blood, and were only punctured a needle to center of the caudate nucleus, but was not injected blood. Preparation of tissue sections and observation of sections florescence and HE staining:Rats of different test groups were anesthetized and the blood vessels were infused, tissues were fixed,then were executed at corresponding time points.Rats in sham operation groups were anesthetized and executed 1 h and 3 d after operation. Brain tissue was separated. Frozen sections were observed under fluorescence microscope and photographed.Green exciting light of 510-550 nm was used to excite for TAMRA-BSA test group, blue light of 460-490 nm was used to excite for FITC test group, and routine HE staining was conducted after fluorescence photographing. The florescent images and HE images of the same site were input into Photoshop CS4, and the images were overlain and analyzed after artificially adjusted.ResultsWhen the hematoma has formed for 1 h, the VRS with lymph retention were broadened in cerebral tissues, peaked at 72h, and edema began to appear in the adjacent cerebral tissues. The inter-vascular space was in balloon change, and edema was obvious in the adjacent cerebral tissues.Seven days later, expanded VRS began to shrink, and lymph retention basically disappeared in VRS 14 days later, and edema of adjacent cerebral tissues also disappeared basically.Edema was not obvious or only mild in brain tissues around the VRS with no apparent lymph retention, lymphatic retention even in the 3 d peak period as well. The extent of brain edema and lymph retention in VRS showed significantly positive correlation.The TAMRA or FITC fluorescent particles were visible in most of the expanded VRS. But not all VRS with TAMRA or FITC fluorescent particles showed lymph retention, a large number of the VRS with TAMRA or FITC fluorescence particle did not show the lymph fluid retention, and the edema surrounding brain tissue was not evident.Meanwhile, some expanded VRS accompanied by brain edema showded non-TAMRA or FITC fluorescent particles. Combined with the positively relationship between the degree of brain edema and the extent of lymph retention in the VRS, lymph retention is the most important reason of brain edema after cerebral hemorrhage.Conclusions1. Compared to intra-cerebral hemorrhage rat model constructed by conventional non-preset canula method, preset canula method can construct stable animal model with cerebral hemorrhage in rat caudate nucleus, the hematoma volume is stable, with less contraflow. It is an effective measure to construct stable cerebral hemorrhage animal model.2. The "piping phenomenon" begins to emerge when the cerebral hemorrhage occurred.3. The "piping phenomenon" of the form components occurs mainly within the VRS.It can spread throughout the central nervous system, while the "piping phenomenon" in nerve fibers is found only in tissues around the hematoma.4. The "piping phenomenon" of formless components extends far beyond distribution of form components. It can occur in the VRS of the whole central nervous system, "The piping phenomenon" in the nerve fibers is mainly seen in the tissue around the hematoma.5. When the formed elements-red blood cells are cracked within hematoma, invisible components can be released, and they spread long-distance along the VRS, which makes the "piping phenomenon" of the formed elements within the hematoma to widen the scope of distribution. 6. The "piping phenomenon" of formless within hematoma reaches a peak when the hematoma formed, and spreads throughout the central nervous system.7. "Piping phenomenon" is one way of the main absorption of the hematoma. Macromolecules precipitated from hematoma are absorbed to the blood mainly by VRS through the deep cervical lymph node.8. "Lymphostatic cerebral edema" in the brain tissues around the hematoma begins to appear after the hematoma formed 1 h,and gets to the peak within 3 days, begins to ease in7 days,14 d later disappears.9. "Piping phenomena" is closely related with the "Lymphostatic cerebral edema". The evolution of the "Piping Phenomenon" in different stages after cerebral hemorrhage leads to dynamic changes of cerebral edema.10. Lymph fluid retention, not hematoma toxic effect, may be the most important reason of the cerebral edema after cerebral hemorrhage,11. The scope of action of toxic substances significantly increases by the "piping phenomenon", and the "piping phenomenon" may be the main mechanism for distant hematoma effect.12. "Piping phenomenon" may result in a hematoma irregular.13."Piping phenomenon" has a relationship with the secondary subarachnoid hemorrhage and secondary intra-ventricular hemorrhage after cerebral hemorrhage.