Investigation On Glymphatic System Change And Its Underlying Mechanism In Glioma

Cerebrospinal fluid(CSF)plays an important role in maintaining tissue homeostasis in the central nervous system.In 2012,the new CSF outflow pathway,“the glymphatic system”was discovered.The glymphatic system is a CSF circulatory system,which consists of cerebral blood vessels and aquaporin 4(AQP4)on the endfeet of astrocytes around the vessels.The glymphatic system involves the para-arterial influx of subarachnoid CSF into the brain interstitium,the exchange of CSF with interstitial fluid(ISF),followed by the para-venous efflux of ISF,which promotes to eliminate harmful solutes in the brain parenchyma.The concept and hypothesis of the glymphatic system and the mechanism in different diseases of the glymphatic system are still incomplete or unknown,which need to be developed and further studied,but these have received more and more attention from researchers and have become a research hotspot or focus.In recent studies of the glymphatic system,the importance of the glymphatic system has been demonstrated in healthy and neurodegenerative disease brains.However,there is limited research on the function of the CSF in brain tumors,especially in the field of glioma.Intracranial hypertension caused by glioma impacts the delivery of chemotherapy drugs via intrathecal injection,which is also associated with the dynamic balance of CSF.The intrathecal injection and the dynamic imbalance of CSF are related with the outflow of CSF pathways.There are three CSF outflow pathways,including the subarachnoid pathway,the perineural pathway and the glympathic system.In previous study,it confirmed that CSF outflow pathways through the subarachnoid pathway and the perineural pathway were blocked in the glioma.However,no one has researched the relationship between glioma and glympathic system.As the glympathic system be the main pathway to eliminate harmful solutes in the brain parenchyma,if the glympahtic system in glioma was blocked,it will give a benefit environment to tumor growth.Therefore,it is urgent to explore the correlation of intracranial pressure(ICP)changes in the glymphatic system and how to explore non-invasive visualization of the glymphatic system in glioma.Thus,in this study,the effect of different tracer infusion rates on ICP in glioma rat model was evaluated using an ICP microsensor;In vivo magnetic resonance imaging(MRI)and ex vivo Evans Blue bright field were used to visualize the glymphatic system in glioma rat model;the relationship between the glymphatic system and AQP4 in glioma rat model by immunofluorescence.Part I Effect of infusion rate on intracranial pressure in glioma rat modelObjective Previously,it was reported that the cerebroventricular injection of a high doses of gadolinium in animals can induce neurotoxic effects,such as focal seizure activity,ataxia,and delayed tremor.To prevent neurotoxicity from a large amount of gadolinium accumulating in the brain in a short time and intracranial hypertension caused by the rapid infusion rate into the cisterna magna,at the same time to obtain the best image information under biosafety conditions,we injected clinical gadolinium contrast agent(Gadobutrol)through the cisterna magna at different infusion rates in orthotopic glioma rat model.An appropriate infusion rate was explored to ensure biological safety,did not cause increased ICP and visualized informational images.Methods C6 glioma cells at a density of 1.0×10~6cells/10μL were implanted into the right striatum to establish an orthotopic glioma rat model.Twelve days after implanting the tumor,the rats were observed the tumor volume by scanning MRI.The pressure probe was inserted into the right lateral ventricle of rats,and the effect of tracer infusion on ICP was evaluated using an ICP microsensor.In the constant-speed group,the ICP was measured at2μL/min by injecting 5μL Gadobutrol via the cisterna magna for 60 minutes.In the variable-speed group,the ICP was measured at each infusion rate(2μL/min,4μL/min,6μL/min and 8μL/min)by injecting 5μL Gadobutrol via the cisterna magna for 25 minutes.The ICP was recorded every 5 minutes.After the operation,the rats were placed in a single cage with a 37°C heating pad to keep warm,and their mental and activity states were observed.An independent-samples t-test was used to compared ICP between the two groups.Paired-samples t-tests were used to compare ICP between each time point in each group.A two-sided p-value<0.05 was considered as statistically significant.Results The ICP of the glioma group was higher than that of the normal group at different infusion rates.In the constant-speed group,there was no significant change in ICP from baseline in either group after the infusion rate of 2μL/min within 60 minutes,and both returned to the normal.In the variable-speed group,at different infusion rates,a progressive significant elevation in ICP was noted in the normal group,especially after the infusion rate of 8μL/min.In the glioma group,the ICP fluctuation was small and not statistically significant compared to baseline,except at 5 and 20 minutes after the infusion rate of 8μL/min.After operation,some rats in two groups had seizure activity and then died.Conclusions The part of this experiment revealed that 2μL/min was a safe injection speed that did not affect the change in ICP and induce neurotoxic effects.Part II Visualization of the glymphatic system in glioma rat modelObjective To investigate whether there is a change in CSF outflow through the glymphatic system in glioma rat model.Methods Twelve days after implanting the tumor,the rats were scanned at before,0.5 h,1 h,2 h,3 h,and 4 h after 5 μL Gadobutrol administration via the cisterna magna by scanning Prisma 3T MRI to observe CSF outflow pathway and the signal intensity in the tumor area.T1-weighted,T2-weighted,and T1-mapping image sequences were acquired.At the same time,using ex vivo Evans Blue imaging,after using the infusion rate of 2μL/min to inject 5 μL 2% Evans Blue into the cisterna magna,rats were sacrificed after 0.5h,1 h,2 h,3 h,and 4 h and perfused with 4% paraformaldehyde for cardiac perfusion.Brains were removed and taken the picture in the bright field.Mann–Whitney U test was used to compare MRI measures between the two groups.For comparisons of the adjacent time points of MRI data within each group,Wilcoxon’s signed-rank test was used.A two-sided p-value <0.05 was considered as statistically significant.Results The T1 mapping coronal MRI data revealed that in the right striatum,the signal intensity of the glioma group was significantly lower than that of the normal group at each time point.Comparison between the contralateral and ipsilateral striatum in the glioma group,the signal of the right tumor was lower than that of the contralateral striatum from 1to 4 h.The T1 mapping sagittal MRI data revealed that contrast agent was obvious around the basilar and anterior cerebral arteries and the olfactory bulbs in the glioma group.Between 1 h and 4 h,the contrast agent gradually spread from the ventral to the dorsal side of the brain,but the signal changes in the tumor area were weak.In the bilateral olfactory bulbs,the signal intensity of the glioma group was higher than that of the normal group at3 and 4 h.In the pineal recess,compared with the normal group,the glioma group was changed no significance by the time.The signal intensity was significantly higher in the glioma group than that in the normal group at 3 and 4 h.Through the observation of Evans Blue ex vivo,in the normal group,the pineal recess,basal cisterna,and surrounding the circle of Willis distributions of Evans Blue were obvious at 1 h.The peak was reached at 2h,and Evans Blue distribution was observed in the branches of the middle cerebral artery in the ventral side of the brain,and then gradually weakened.In the glioma group,Evans Blue appeared earlier.The pineal recess,basal cisterna,and surrounding the circle of Willis distributions of Evans Blue were obvious at 0.5 h,and then gradually weakened.The change of Evans Blue in the pineal recess of the glioma group was not obvious over time.Regardless of whether observations of the brain were made dorsally or ventrally,the distribution of Evans Blue around the middle cerebral artery was not obvious in the tumor area compared with that in the contralateral side.Conclusions The part of this experiment revealed that CSF outflow pathways through the glymphatic system were blocked in glioma rat model.At the same time,in the glioma rat model,CSF outflow pathways through the subarachnoid pathway and the perineural pathway were blocked,which were consistent with the results reported in the previous literature.Part III Relationship between the glymphatic system and aquaporin 4 in glioma rat modelObjective To explore the reason for the obstruction of CSF via the glympahtic system in the glioma rat model,our study aims to clarify the relationship between the glymapthic system and AQP4 in the glioma rat model.Methods The brains,which were injected with Evans Blue,were sectioned at 10 μm thickness in the coronal plane using a freezing microtome,and were labeled with glial fibrillary acidic protein(GFAP)and AQP4.The un-injected rat brains were sectioned at 4μm thickness in the coronal plane after deparaffinization and rehydration.The slices were stained with GFAP,anti-alpha-smooth muscle actin(α-SMA),CD34 and AQP4,and observed using a confocal microscope and photographed for quantitative analysis.Mann–Whitney U test was used to compare average fluorescent intensity between the two groups.A two-sided p-value <0.05 was considered as statistically significant.Results In the glioma group,the fluorescence intensity of GFAP in the tumor area was significantly higher,whereas the fluorescence intensity of AQP4 was significantly weaker.The fluorescence intensity of Evans Blue in the tumor area was also reduced.Using high-power resolution,we found that AQP4 around the GFAP-labeled astrocyte was decreased,and Evans Blue surrounding AQP4 was also decreased.In the tumor area,irrespective of CD34+α-SMA+ arteries or CD34+α-SMA-veins,AQP4 expression surrounding the vessels was weak.Data indicated that the number of CD34+α-SMA+ arteries in the tumor area were more than in the contralateral area.In contrast,the number of CD34+α-SMA-veins in the tumor area were less than in the contralateral area.Conclusions The part of this experiment revealed that the impediment of CSF drainage through the glymphatic system in glioma rat model might be related to the decreased expression of AQP4 on the endfeet of astrocytes around the vessels.