| Perivascular spaces (PVS) was named by by R. Virchow (the German pathologist) and C.P.Robin (the French biology and organizations scientist) more than a century ago, named Virchow-Robin space (VRS) later. It was also called perivascular lymph space. VRS was separated from the blood vessels and surrounding brain tissue in the nervous system, as the normal anatomical structure, becoming a research hotspot.Although, there is no real lymphatic vessels embedding endothelial cells in the central nervous system, of specific lymphatic drainage pathway in brain is specifically existesting, acting a very important role for the maintenance of physiological function. A series of lymphatic stasis of the disease could be induced by blocking the lymphatic drainage pathways in brain, which is called lymphatic encephalopathy (Lymphostatic Encephalopathy, LE). The VRS within the brain tissue is a major component of the lymphatic system, making up the Prelymphatic System, which is one of the ways, the spread of disease.Intracerebral hemorrhage (ICH) is a common disease, harmful seriously to human health. There were many "spot bleeding" around the organization of hematoma after ICH. These "spot bleeding" lesions often located along the VRS and the nerve fibers from the high pressure to the low. The "spot bleeding" can congest the VRS, causing the remote VRS. Whether the monocyte-macrophage system was activated after ICH? In recent years, more and more scholars begin to pay attention to the role of VRS. What is the connection between the VRS and the immune response in the CNS? And what is the kind of specific lymphatic circulation path? But so far, there is no clear evidence about that. For comparative studies, we observed the Vichow-Robin Spaces after bilateral ligation and removal of cervical lymph nodes,which blocked the cerebral lymphatic drainage. Then we made the rat model of vasogenic brain edema, which was leading lymph formation. We discussed the mechanisms of the dilated VRS in different angles and the relationship between VRS and immunity.Ⅰ. A rat Model of Focal Lymph Encephalopathy Constructed with Cerebral Superficial Artery Semi-ligation and the Pathological Study of Dilated VRSObjectiveIn this article, via cerebral superficial artery semi-ligation surgery, we constructed a focal lymphatic encephalopathy rat model which is repeatable and stable without affecting the brain function. We observed the changes of Virchow-Robin Space (VRS) under the microscope.Materials and methods40 SD male rats (Experimental Animal Center of Southern Medical University offered), Weighing 250±10 g, randomly divided into 24h,48h model group group, 24h,48h sham group,10 in each group.Another 5 healthy male SD rats were the 48h model group which perfused the Cerebral vessels with Trypan blue. After intraperitoneal anesthesia with 10% chloral hydrate (0.4ml/100g), the body fixed for prone position, parietal skin preparation, disinfection, median longitudinal scalp incision 2cm, exposure through corrosiving skull periosteum with 30% hydrogen peroxide,we begun to location:next the anterior fontanel, avoiding the superior sagittal sinus. We drill a hole with the dental drill, diameter 3-5mm, to the dural surface, and opened the dura.Then the rats were moved to the stereo microscope, exposing the brain surface artery. We blocked the VRS, via semi-artery ligation with 3/8 nylon line, which the blood vessels remained patent, not affecting the distal blood flow. The scalp was sutured after operation. The sham group only isolated rat brain surface artery without a half ligation operation. All rats were injected with tetramethyl rhodamine-bovine serum albumin (BSA-TAMRA) 10μl in homonymy. EEG monitoring (elica) was used through the operation in rats. We could compare the differences whether the cerebral ischemia and other changes of brain function. The brain of all rats were perfused and fixed. We observed the brain tissue in the fluorescence microscope after frozen sections and in the ordinary light microscope after H-E staining.ResultsEEG monitoring showed that the operation does not change the brain function. Under the light microscope, the blood-brain barrier was not damaged in the model group through trypan blue perfusion. Under the fluorescence microscope, a large number of BSA-TAMRA gathered in the cortex and subcortical white matter within the VRS under the semi-ligation. Many VRS were expanded, distributing in the subcortical white matter commonly. The brain tissue around the dilated VRS was loose,and pale. Statistical analysis:At 24h and 42h, the expanded VRS numbers in model group (24h:6.60±2.76; 48h:6.90±2.33) were more than the sham group, significantly increased (P=0.001); there was no significant difference of the numbers between the 24h and 48h model group(F=0.338,P=0.564); statistics analysis to the cross-sectional area of dilated VRS:homogeneity of variance test, F=8.460, P= 0.001, Heterogeneity of variance. Approximate F test using Welch method, F= 40.913, P= O.OOO.There was significant differences, needing multiple comparison. The cross-sectional areas were significantly increased in the model groups (24h: 4.09±2.98 um2; 48h:4.08±1.37 um2; F=40.913, P=0.000); model group, and there was no significant difference between 24h and 48h model group (P= 1.000).Ⅱ.Pathological Study of VRS after Intracerebral Hemorrhage Causing the Focal Lymph EncephalopathyObjectiveIn order to confirm whether the immune system was activated after intracerebral hemorrhage (ICH), and observe the pathological changes of VRS, the relationship between immune response after intracranial hemorrhage and VRS, we developed an approach by the secondary pre-injection of autologous arterial blood to build ICH model in rats. It caused the brain regional lymph circulation disorder. We observed via immunohistochemistry and immunofluorescence markers (CD68 and CD4 positive cells). Under the ordinary light microscope or fluorescence microscope, we observed the positive cells in brain. In this study,we also used the intracranial injection the mixture of autologous blood and BSA-TAMRA and the individual injection of BSA-TAMRA, to further observation of the VRS path of intracranial Lymph circulation and the specific characteristics.Materials and methodsSD male rats, weighing 250±10 g (offered by the Experimental Animal Center of Southern Medical University), were maintained at normal laboratory temperature, free of water consumption. 1. Intracranial injection of autologous blood:20 rats were randomly divided into model group and control group,10 in each. After depth of anesthesia by intraperitoneal injection of 10% chloralhydrate (0.4mL/100g), the rats were fixed in the stereotaxic apparatus, and was drilled a hole at the needle point, diameter 1mm, to the hard meningeal surface.24G intravenous catheter was placed into the center of the caudate nucleus (respect to bregma:1 mm anterior,3 mm left lateral,6 mm depth). After 24h, the rats were anesthetized again, and 20μL blood was drawn from femoral artery with a micro syringe. In model group, 10μL arterial blood was slowly injected (10μL/min) into the caudate nucleus.8 minutes later, the remaining 10μL of blood was injected with the same speed.. The control group rats were injected with normal saline by the same way.2. Preparation and intracranial injection of the mixture of autologous blood and BSA-TAMRA:by the same approach, we injected the mixture into the left brain. The group contained 10 SD male rats and all rats were killed after 24h.3. Intracranial injection of BSA-TAMRA:BSA-TAMRA was injected into the left hemisphere of rats under the stereotactic apparatus. The rats were randomly divided into three sub-groups:24h group,48h group, and 72h group, with 10 rats in each group. The rats were separately executed at 24h,48h,72h after operation.All rats were put into intracardiac perfusion of 4% paraformaldehyde under anesthesia. Then the brain tissues were prosecuted frozen section. With the image analysis system Mias 2000, we measured the average cross-sectional area of VRS in each rat brain tissues (10 cross-sectional areas for each rat). With the SPSS 13.0 statistical software, single factor analysis of variance, homogeneity of variance test first, the multiple comparisons were carried on. Results1. Under the fluorescence microscope, CD4, CD68 positive cells in the model group (6.50±2.46) were significantly increased compared with the control group (2.80±1.87个) (t=3.783,P=0.001), clustering within the VRS. H-E staining of rat brain tissue showed that, there were large areas of bleeding lesions in brain tissue, spreading to the distal along the VRS and around the vessels. CD68 immunohistochemistry of brain tissues showed the same result.2. The mixture of autologous blood and BSA-TAMRA could migrate and diffuse along the VRS:Under the fluorescence microscope (WG, excitation wavelength 510-515nm, absorption wavelength 590nm), the mixture could be seen gather in the VRS (red fluorescence). After taking fluorescent film, the ordinary light image was taken umder the microscope in the same field, and overlapped on the fluorescence image. After H-E staining, from the brain blood vessels to the brain surface, the mixture was along the blood vessels more intensively, or even filled blocking, causing VRS expansion.3. BSA-TAMRA migration path observed in the brain:In 24h group, the fluorescence mainly in the left basal ganglia and cortex. In 72h 48h group, and the fluorescent particles are more dispersed within the VRS. A large number of BSA-TAMRA particles gathered in the brain ventricle and the VRS of meningeal blood vessels. In addition, this study also found that a large number of BSA-TAMRA move to bilateral cervical lymph node. Ⅲ. A rat Model of Whole Brain Lymph Encephalopathy Constructing and the Pathological Study of Dilated VRSObjectiveVia ligation and removal bilateral cervical lymph nodes, we constructed a rat model of whole brain lymph encephalopathy, causing lymphatic returning obstruction. After immunofluorescence antibody labeled monocytes (CD68), we observed under the fluorescence microscope, whether the Monocyte-macrophage system was activated.we also detected the relationship between the traffic pathes and the VRS. Further, we could explore the function of VRS and its relationship with the immune response.Materials and methods40 healthy male SD rats (Experimental Animal Center of Southern Medical University), weight 250±10g, were randomly divided into 24h/48h model group, 24h/48h sham operation group (isolated superficial cranial arteries but no half-ligation), with 10 rats in each group. All rats were raised with standard diet, free access to water, room temperature 22-25℃.We constructed the rat model of whole brain lymph encephalopathy by ligation and removal the bilateral cervical lymph nodes. The control group rats needed not ligation and removal of lymph nodes. Other steps were the same as the model group.Put the prepared organizations under the frozen section machine (LEICA CM 1850), the temperature at-20℃(30min). The brain tissues were prosecuted frozen section. The brain tissue slices were stained by immunofluorescence. Then we put them under the fluorescence microscope (OLYMPUS BX51) (WG, excitation wavelength 510-550nm, absorption wavelength 590nm). In the same field, we took the fluorescent film and ordinary photo to overlap. Last, after H-E staining, we observed under light microscope and took photo, and counted the numbers of dilated VRS. With the image analysis system Mias 2000, we measured the average cross-sectional area of VRS in each rat brain tissues (10 cross-sectional areas for each rat). Statistical analysis was carried on with the SPSS 13.0 statistical software. P <0.05 as significant difference.ResultsIn 24h model group, under the light microscope, we observed that there were large numbers of dilated VRS, around the blood vessels in the cortex and Basal ganglia. The VRS showed round or strip, containing transparent tissue fluid and cells. The numbers and cross-sectional areas of the expanded VRS (6.17±2.49) were significantly higher than control group (0.39±0.28) (F=107.019, P=0,000).Under the fluorescence microscope, CD68 positive cells gathered in the VRS showing the red fluorescent light. In the 48h group, we found the same result (14.90±5.55) (F=53.588,P=0.000)Ⅳ. Pathological Study on the Vichow-Robin Spaces in an Animal Model of Vasogenic Brain Edema after Hypertensive EncephalopathyObjectiveIn this article, via Pathological technique, under the microscope, we observed the Vichow-Robin spaces in the animal model of vasogenic brain edema after hypertensive encephalopathy, and discussed the mechanisms of the dilated VRS. Materials and methods20 SD rats (Experimental Animal Center of Southern Medical University), weight 250+10g, were randomly divided into model group and control group(n=10). Offered:standard diet, free access to water, raising temperature 22-25℃. In model group, all rats were injected with phenylephrine in enterocoelia(6.0mg/kg,q8h, three times in all). While control group rats were injected with saline. After 48h, all rats were executed under anesthesia with chloraldurat. The brain tissues were sliced into sections with a freezing microtome after perfusion fixation and observed under light microscope after H-E staining. The features of VRS were observed and quantified by Mias 2000 image analysis system.Using SPSS 13.0 statistical software to compare the number of VRS between the two groups and the expansion of cross-sectional area. Measurement data with a mean±standard deviation (X±s) indicated. P＜0.05 as significant difference.ResultsUnder the optical microscope, model group rats showed the apparently dilated VRS. Tissues, around VRS were pale, stained crumbly and lightly. Some even appeared sponge-like edema, while the control group was normal. Contrast with the control group (1.40±1.26) (4.54±2.35um2), the munbers of dilated VRS (7.80±2.53) and cross-sectional area (0.31±0.27um2) in modol groupwere notable increasing (t=5.642, P=0.000).Conclusions1. Cerebral superficial artery semi-ligation surgery can construct a focal lymphatic encephalopathy rat model which is repeatable and stable without affecting the brain function and maintain the integrity of BBB.2. Focal lymphatic encephalopathy can lead the cortical and subcortical VRS limited expansion, appearing round, oval, linear, etc., located in the cortex and subcortical district.3. Hematoma can expand along the VRS to the remote, pluging the VRS, causing the lymphatic retention, proximal VRS expansion.4. Monocyte-macrophage system is activated after ICH, resulting immune response.5. A large number of macrophages gather in the VRS, following along with the the intracranial lymph circulation, migrating to meningeal VRS and brain ventricle.6. VRS are the location and pathway in the CNS—activating immune response and cleaning the foreign substances.7. A large number of fluorescein particles gather in the VRS, following along with the the intracranial lymph circulation, migrating to meningeal VRS and brain ventricle.they can be recycled to the bilateral cervical lymph nodes ultimately.8. The intracranial lymph circulation is interlinked with bilateral cervical lymph nodes.9. Whole brain lymphatic encephalopathy model cause cerebral lymphatic drainage blocked, brain swelling. A large number of VRS expanded, scattering in all brain.10. The cross-sectional area and the numbers of dilated VRS are significantly increasing after whole brain lymphatic return obstruction,which is the mechanism of VRS expanding.11. Whole brain lymphatic encephalopathy could activate the monocyte-macrophage system, within the VRS, around the blood vessels.12. Hypertensive encephalopathy can cause the blood-brain barrier damaged, the vascular permeability increased, lymph increased, the vasogenic brain edema formed at last. 13. Vasogenic brain edema after hypertensive encephalopathy brings aboutthe expansion of many VRS and the cross-sectional area increasing significantly.It is one of the mechanisms for the expansion of VRS. |
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