Application of related products of gliolymphatic system in preparation of products for diagnosing or treating sepsis encephalopathy

Abstract

The invention belongs to the field of biological medicine, and discloses application of a product related to a gliolymphatic system to preparation of a product for diagnosing or treating sepsis encephalopathy. It is found for the first time that the gliolymphatic system can effectively diagnose sepsis encephalopathy, cognitive impairment, neuroinflammation and astrocyte A1 type activation severity of the sepsis encephalopathy. The invention also finds that promotion and improvement of gliolymphatic system reflux are effective therapeutic targets for sepsis encephalopathy. The invention provides a new thought for preparing diagnosis products and treatment products (such as adenovirus products capable of coding and synthesizing vascular endothelial growth factor C) of sepsis encephalopathy, and has a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to the application of related products of the glial lymphatic system in the preparation of products for diagnosing and treating septic encephalopathy. Background Art

[0002] Sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection and is one of the leading causes of death in critically ill patients. Sepsis is often accompanied by short-term diffuse brain dysfunction and long-term irreversible cognitive impairment, namely sepsis-associated encephalopathy (SAE). Clinically, SAE is characterized by changes in behavior, cognition, arousal, and consciousness, and a considerable proportion of patients have long-term cognitive dysfunction, which seriously affects daily life and increases the risk of death. The diagnostic criteria for SAE lack specificity and are an exclusionary diagnosis. It is necessary to exclude encephalopathy caused by drug use and other causes. The imaging manifestations of SAE are not specific and often require a combination of clinical history and laboratory tests to exclude other central direct infections before diagnosis. However, the prognosis of the disease is poor, and early diagnosis and active treatment are of great significance to improving the prognosis of the disease.

[0003] Brain metabolic waste removal is essential for maintaining brain homeostasis, lipid transport, and immune surveillance throughout the life cycle. In the past, it was believed that cerebrospinal fluid (CSF) plays a role similar to the lymphatic system in this process. However, CSF diffuses very slowly in brain tissue (it takes 100 hours to diffuse 1 cm), which contradicts the efficient metabolic waste removal system that matches the high metabolic activity of neurons and cannot meet the high demand of synaptic transmission for brain homeostasis. In 2012, it was first discovered that there is a glial lymphatic system (Glymphatic System) in the brain parenchyma, which is composed of the paravascular space between the vascular end feet of astrocytes and the vascular adventitia they are wrapped in. With the pulsation of the arteries, CSF in the subarachnoid space enters the deep brain parenchyma along the para-arterial perivascular space, and rapidly enters the brain parenchyma under the promotion of rapid water transport by aquaporin 4 (AQP4) distributed on the end feet of astrocytes, and then flows back through the paravenous perivascular space, and finally merges into the dura mater lymphatic vessels, thereby being excreted out of the brain. Since the foot processes of astrocytes are not tightly connected but have large gaps, soluble waste proteins and protein aggregate-like metabolites can be discharged along with CSF through this pathway. This overall flow of fluid from the para-arterial perivascular space to the para-venous perivascular space is the basis for the clearance function of the glymphatic system, and the clearance process through this pathway is non-specific and very efficient. Therefore, the brain glymphatic system realizes direct and continuous drainage between the brain parenchyma and the central perilymphatic system, which is beneficial to the maintenance of the homeostasis of the brain's internal environment. The glymphatic system is regulated by many factors, such as the polar distribution of AQP4 around blood vessels, respiratory movement, arterial pulsation and arteriosclerosis, wakefulness and calmness, and dura mater lymphatic vessels. Studies have found that a variety of neurological diseases including traumatic brain injury, sleep disorders, ischemic stroke, multiple sclerosis, Parkinson's disease, Alzheimer's disease, etc. have glymphatic system transport disorders and metabolic waste accumulation, leading to a series of cognitive dysfunctions. However, there are currently no studies at home and abroad reporting on the role and function of the glymphatic system in sepsis and septic encephalopathy. Summary of the invention

[0004] The technical problem to be solved by the present invention is to overcome the deficiencies and defects mentioned in the above background technology and provide an application of a glial lymphatic system in the diagnosis and treatment of septic encephalopathy.

[0005] In order to solve the above technical problems, the technical solution proposed by the present invention is: A reagent for detecting reflux disorders of the brain gliolymphatic system is used in the preparation of a product for diagnosing septic encephalopathy.

[0006] In the above application, preferably, the septic encephalopathy is septic encephalopathy simulated by a cecal ligation and puncture model.

[0007] Preferably, the reagent for detecting brain gliolymphatic system reflux disorder includes the magnetic resonance paramagnetic contrast agent Gd-DOTA for injection into the cisterna magna of the brain.

[0008] Based on a general inventive concept, the present invention also provides an application of a product for promoting the reflux of the glial lymphatic system in the preparation of a product for treating septic encephalopathy.

[0009] In the above application, preferably, the septic encephalopathy is septic encephalopathy simulated by a cecal ligation and puncture model.

[0010] Preferably, the product that promotes the reflux of the glial lymphatic system is vascular endothelial growth factor C.

[0011] Preferably, the product promoting the reflux of the glial lymphatic system is a gene expression product that can encode the synthesis of vascular endothelial growth factor C.

[0012] Preferably, the gene encoding vascular endothelial growth factor C is as shown in SEQ ID No.1.

[0013] Preferably, the expression vector of the gene expression product is an adenovirus.

[0014] Compared with the prior art, the present invention has the following beneficial effects: The present invention discovered for the first time that the glial lymphatic system can effectively diagnose septic encephalopathy, as well as the severity of cognitive dysfunction, neuroinflammation, and astrocyte A1 activation. The present invention also discovered that promoting and improving the reflux of the glial lymphatic system is an effective therapeutic target for septic encephalopathy. The present invention provides a new idea for preparing diagnostic products and therapeutic products for septic encephalopathy (such as adenovirus products that can encode synthetic vascular endothelial growth factor C), and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0016] Figure 1 These are 9.4T MRI scan images of the mouse brain after Gd-DOTA was injected into the medulla oblongata to simulate metabolic waste in the brain; A is the Bruker 9.4 T MRI small animal cranial imaging scanning system; B is the representative original MRI images of the sham operation (control) group and the cecal ligation and puncture (septic encephalopathy) group based on voxel morphological analysis (coronal, sagittal, and transverse planes, respectively).

[0017] Figure 2 Figure 1 is a quantitative analysis of longitudinal relaxation time imaging (T1 mapping) in various brain regions after Gd-DOTA was injected into the medulla oblongata of mice to simulate metabolic waste in the brain; A is a representative MRI voxel image (coronal and sagittal cross-section) of the sham operation (control) group and the cecal ligation and puncture (septic encephalopathy) group; B is the mean regional T1 value (Mean regional T1) of various brain regions of mice: hippocampus, cerebellum, hypothalamus, olfactory bulb, thalamus, superior colliculi, interior colliculi, basal forebrain septum. The statistical results of T1 value showed that the larger the T1 value, the more Gd-DOTA accumulated in the brain, which meant that the clearance function of the glymphatic system was worse. A significant increase in T1 value was observed in all brain regions of mice in the septic encephalopathy group, and the differences were statistically significant (cerebellum **p<0.01; hippocampus **p<0.01; hypothalamus ***p<0.001; olfactory bulb ***p<0.001; thalamus *p<0.05), indicating that the clearance efficiency of Gd-DOTA injected into the cerebrospinal fluid of mice with septic encephalopathy was significantly reduced, and there was a serious disorder of brain glymphatic system transport.

[0018] Figure 3 is the result of co-localization immunofluorescence of GFAP and AQP4 in brain slices; A is the immunofluorescence image of brain slices, blue is the cell nucleus (DAPI), green is AQP4, red is GFAP, the white box is the local part of AQP4-positive astrocytes before magnification, Zoom is the co-localization of AQP4 and GFAP after magnification, scale bar = 100μm; B is the statistical average fluorescence intensity data of AQP4 localized on GFAP-labeled astrocytes (n=6 mice / group, Student's t test: Sham vs. CLP; *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.001); C is the correlation analysis of T1 values ​​of the same batch of mice and the average fluorescence intensity data of AQP4 localized on GFAP-labeled astrocytes.

[0019] Figure 4 The open field test was used to test the anxiety-like behavior of mice (n=6 mice / group); A is a representative open field test picture of the sham operation group and the cecal ligation and puncture group, and B is the correlation analysis between the moving distance of the central area and the T1 value.

[0020] Figure 5This is a novel object recognition experiment to test the memory function of mice (n=6 mice / group); A is a representative novel object experiment picture of the sham operation group and the cecal ligation and puncture group, and B is the correlation analysis between the novel object residence time and the T1 value.

[0021] Figure 6 The Y-maze experiment was used to test the memory function of mice (n=6 mice / group); A is a representative Y-maze experiment picture of the sham operation group and the cecal ligation and puncture group, and B is the correlation analysis between the percentage of alternation in the novel arm and the T1 value.

[0022] Figure 7 Figure 3 is a cross elevated maze test to test anxiety-like behavior in mice (n=6 mice / group); A is a representative cross elevated maze test picture of the sham operation group and the cecal ligation and puncture group, and B is the correlation analysis between the open arm residence time and the T1 value.

[0023] Figure 8 Figure 2 is the immunofluorescence result of brain slices with GFAP marking astrocytes and C3 marking A1 activated astrocytes; A is the immunofluorescence image of brain slices, blue is the nucleus (DAPI), green is GFAP, red is C3, the white box is the magnification of AQP4-positive astrocytes, Zoom is the co-localization of C3 and GFAP after magnification, scale bar = 100μm; B is the statistical average fluorescence intensity data of C3 located on GFAP-labeled astrocytes (n=6 mice / group, Student's t test: Sham vs. CLP; *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.001); C is the correlation analysis of T1 values ​​of the same batch of mice and the average fluorescence intensity data of C3 located on GFAP-labeled astrocytes.

[0024] Fig. 9 The following are the expressions of vascular endothelial growth factor C (VEGF-C) in the mouse brain and its transport through the glial lymphatic system after injection of adenovirus overexpressing VEGF-C; A is a protein immunoblot verification of the successful construction of AVV-VEGF-C overexpression in brain tissue, and B is a 9.4T magnetic resonance imaging image of the transport and drainage efficiency of cerebrospinal fluid in the deep cervical lymph nodes and glial lymphatics of mice.

[0025] Fig.10 The results of the anxiety-like behavior experiment in the open field test of mice in the sham operation group and the cecal ligation and puncture group after pre-injection of adenovirus overexpressing vascular endothelial growth factor C (VEGF-C) (n=6 mice / group).

[0026] Fig.11The results of the Y-maze test on the memory function of mice in the sham operation group and the cecal ligation and puncture group after pre-injection of adenovirus overexpressing vascular endothelial growth factor C (VEGF-C) (n=6 mice / group).

[0027] Fig.12 These are the results of a novel object recognition experiment testing the memory function of mice in the sham operation group and the cecal ligation and puncture group that were pre-injected with adenovirus overexpressing vascular endothelial growth factor C (VEGF-C) (n=6 mice / group).

[0028] Fig.13 The results of the cross-elevation test on anxiety-like behavior of mice in the sham operation group and the cecal ligation and puncture group after pre-injection of adenovirus overexpressing vascular endothelial growth factor C (VEGF-C) (n=6 mice / group).

[0029] In the above figures, Sham represents the sham operation (control) group, and CLP represents the cecal ligation and puncture simulation sepsis mouse model encephalopathy group. DETAILED DESCRIPTION

[0030] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively and meticulously below in conjunction with the accompanying drawings and preferred embodiments of the present invention, but the protection scope of the present invention is not limited to the following specific embodiments.

[0031] Unless otherwise defined, all professional terms used below have the same meanings as those generally understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention.

[0032] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0033] The glial lymphatic system described in the present invention is a cerebrospinal fluid-brain tissue fluid rapid exchange flow system mediated by aquaporin-4 on astrocyte terminal feet, and is composed of four parts: periarterial space, astrocyte terminal feet, neural tissue interstitium and perivenous space.

[0034] The magnetic resonance paramagnetic contrast agent Gd-DOTA described in the present invention has the following information: Chinese name: gadoteric acid; Chinese alias: 2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl] gadolinium acetate, English name: Gadoteric acid, Gd-DOTA, DOTA-GD; English alias: 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid, gadolinium complex; 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, gadolinium salt (1:1); CAS No.: 72573-82-1; Molecular formula: C 16 H 28 GdN4O8.

[0035] Example: 1. Experimental method.

[0036] 1.1 Establishment of a mouse model of septic encephalopathy.

[0037] Cecal ligation and puncture (CLP) model: C57 mice were fasted for 12 hours before surgery and had free access to water. Anesthesia was maintained by inhalation of isoflurane, and the mice were fixed in the supine position. The abdomen was disinfected, and a 2 cm longitudinal incision was made along the midline of the abdomen to separate the cecum. The root of the cecum was ligated below the ileocecal valve with a 3.0 silk thread, and the cecum was punctured with an 18-gauge needle to squeeze the cecum to prevent feces from overflowing. The cecum was then returned to the abdominal cavity, and the abdominal wall incision was sutured layer by layer. The mice were placed on a heating pad to keep warm until they woke up and could move normally, and 50 mL / kg body weight of normal saline was injected subcutaneously to prevent shock.

[0038] 1.2 Injection of the paramagnetic contrast agent Gd-DOTA into the cerebellar spinal cistern.

[0039] In order to objectively and quantitatively study the function of the glymphatic system for brain waste metabolism, MRI paramagnetic contrast agents can be injected into CSF ​​through the cerebral cerebellar spinal cistern as a substitute for "brain waste solutes". The entry of CSF tracers from the periarterial space into the brain tissue is called the "inflow" of the glymphatic system, while the outflow of tracers from the brain tissue is called the "clearance" or "excretion" of the glymphatic system. MRI imaging is used to describe its key physiological processes. This experiment uses a paramagnetic contrast agent gadoteric acid with a relative molecular mass of 652.7. Because of its special size, it can freely pass through the perivascular space outside the blood vessels, is distributed in the extracellular space fluid, and cannot pass through the blood-brain barrier to enter the blood and cell membranes to enter the cells. It is a perfect substitute for soluble brain waste solutes. Mice were fasted for 12 hours before surgery and had free access to water. Anesthesia was induced by 4% isoflurane gas and fixed in a brain stereotaxic instrument. Routine disinfection, 2% lidocaine anesthesia, incision of skin and subcutaneous tissue, exposure of cerebellar spinal cistern, microinjection syringe inserted 2mm, using a 30 Ga needle connected to a micropump and catheter inserted 2mm, 1:37 paramagnetic contrast agent Gd-DOTA injected at 1.5uL / min, total dose of 30uL. The needle was retained for 2min, and the skin was glued with absorbable glue.

[0040] 1.3 MRI sequence and image acquisition.

[0041] Magnetic resonance data were acquired on a 9.4 Tesla (T) magnetic resonance imaging system (Bruker, 9.4-Tesla Scanner) with a GA-9S-HP gradient coil. The magnetic field gradient was controlled by the Bruker Advance III console Paravision 6.1 software (Bruker Bio Spin, Billerica, MA, USA). During the scan, foam padding was used to restrict the movement of the head to reduce the noise of the scan data, and a magnetic-resistant vital sign monitor (SAInstruments, Inc., Stony Brook, NY, USA) was used to continuously monitor physiological parameters including respiratory rate, body temperature, and heart rate. The body temperature of the mice was maintained between 36.5 and 37.5°C using a heating pad.

[0042] B1 relaxation-enhanced rapid acquisition images were acquired using the following parameters: RF repetition time (TR) = 10000 ms, echo time (TE) = 22 ms, flip angle (Flip Angle) = 70°, 140°, RARE factor = 4, imaging field of view (FOV) = 256 × 256 mm 2, Slice Thickness = 1.2mm, Slice Gap = 1mm, 32 layers, scanning time 3min. T1-weighted images (structural images) were obtained using spoiled gradient recall sequence with the following parameters: RF repetition time (TR) = 15ms, echo time (TE) = 2.5ms, 4.5ms, flip angle (FlipAngle) = 2°, 5°, 10°, 15°, 20°, 30°, imaging field of view (FOV) = 256 × 256 mm2, slice thickness (Slice Thickness) = 1.2mm, slice gap (Slice Gap) = 0mm, voxel size (Voxel Size) = 1×1×1mm 3 , scan time = 2.5min.

[0043] 1.4 Processing and analysis of MRI images.

[0044] The magnetic resonance data were processed and analyzed by MATLAB 2018b (Mathworks, MA, USA) code, SPM 12 (statistical parametric mapping software) software package and FSL software.

[0045] Magnetic resonance data preprocessing: The original 2DSeq format of the relaxation-enhanced rapid acquisition sequence and spoiled gradient echo sequence obtained for each experimental rat was converted into the NIFTI format using the MATLAB program code Bruker_batch.m provided by Bruker.

[0046] Correction of spatial non-uniform distribution of RF emission (B1+): Due to the non-uniform distribution of spatial B1+, there is an error between the effective flip angle (θe) and the theoretical flip angle (θn), and the relationship between them is θe = αθn, where α is the correction factor. The correction factor can be calculated using the dual-angle (70°, 140°) RARE sequence images, and the formula 1 is as follows: …[Formula 1]; Where θ1 is 70°, I θ =70 and I θ=140 respectively flip angle 70° and 140° magnetic resonance signal intensity. The calculated correction factor α can be used to calibrate the spatial magnetic field of all SPGR images to avoid the influence of B1+ spatial inhomogeneity on longitudinal relaxation time quantitative imaging. α is used to correct VFA-SPGR images.

[0047] Longitudinal relaxation time quantitative imaging processing: The relationship between the 3D variable flipangle spoiled gradient echo sequence (VFA-SPGR) magnetic resonance signal (S) and repetition time (TR) and longitudinal relaxation time quantitative (T1) is shown in formula 2: …[Formula 2]; Where S0 represents the proton density signal, and dividing both sides of Formula 2 by sinθe yields Formula 3: … [Formula 3]; From formula 3, it can be seen that S / sinθe and S / tanθe are in a linear relationship, and the slope k and intercept b are expressed by formulas 4 and 5 respectively: …[Formula 4]; … [Formula 5]; The slope k and intercept b can be calculated by linearizing and unweighted least squares fitting the six VFA-SPGR magnetic resonance signals (S) obtained by scanning. T1 and S0 can be obtained by solving equations 4 and 5, as shown in equations 6 and 7: …[Formula 6]; … [Formula 7]; Briefly, 3D T1 maps and 3D proton density weighted (PDW) anatomical images were calculated using 3D VFA-SPGR signal linearization and unweighted least squares fitting according to Equations 1-7.

[0048] Longitudinal relaxation time quantitative imaging analysis: Using 3D PDW as the anatomical image, the ITK-SNAP (version 3.8.0 www.itksnap.org) software was used to manually draw the region of interest (ROI) of the rat brain in the glymphatic system, including the hippocampus, hypothalamus, thalamus, brain stem, cerebellum, midbrain, olfactory bulb, and basal forebrain. The relaxation time quantitative images were preprocessed to exclude T1 values ​​greater than 5000 ms, and then the ROIs were overlaid on the processed T1 map. The mean and variance of T1 of each ROI were extracted using the self-developed MATLAB code as the inflow of the glymphatic system in the region. The smaller the T1 value, the greater the concentration of the paramagnetic contrast agent Gd-DOTA, that is, the greater the inflow of the glymphatic system.

[0049] Analysis of Gd-DOTA concentration in the glymphatic system: The relationship between the contrast agent Gd-DOTA concentration and the longitudinal relaxation time (T1) is shown in the Solomon-Bloembergen equation: … [Formula 8]; Among them, T1 t is the T1 value without contrast agent, T1 post represents the T1 value after injection of contrast agent Gd-DOTA, n Gd is the contrast agent Gd-DOTA concentration (mM), C Gd is the constant of the magnetic resonance T1 value at a field strength of 9.4T changing with the concentration of the contrast agent Gd-DOTA, and the value is 2.69 mM -1 s -1 . Formula 8 is transformed to obtain T1post: …[Formula 9]; …[Formula 10]; In a small concentration range, the increase in Gd-DOTA concentration is proportional to the decrease in T1 value. Therefore, we can directly use the size of the T1 value in each brain region to represent the distribution of the contrast agent in the brain. The formula is as follows: for formula 10, the concentration of contrast agent Gd-DOTA n Gd The derivative is: … [Formula 11]; … [Formula 12]; T1 post With n Gd The slope is a constant -T1t 2 C Gd , that is, the increase of contrast agent concentration is directly proportional to the decrease of T1 value. At the same time, the feasibility of this process (Formula 12) is simulated using the preliminary experimental results.

[0050] 1.5 Animal behavior.

[0051] All behavioral experiments in the experiment of the present invention were conducted between 09:00 and 17:00. The experimenter was exposed to the mice 7 days or more before the formal experiment to make the mice familiar with the experimenter. Three hours before the experiment, the mice were placed in the behavioral laboratory to allow them to adapt to the environment of the behavioral room. The experimenter could not make too much noise during the test and could not have strong odors on his body, because noise and odors may cause anxiety stimulation to the mice and affect the behavioral results.

[0052] (1) Open field test (OFT).

[0053] Experimental steps: Take the test mouse out of the cage and carefully place it in a corner of a 42cm (length) × 42cm (width) × 42cm (height) untopped experimental box from the same position and angle. The operator leaves the behavioral laboratory and lets it explore freely for 10 minutes. The whereabouts of the mouse are recorded by a video tracking system, and the time the mouse stays in the middle area and the number of times it crosses the central area are counted. After the 10-minute test, the mouse is returned to the cage. After each mouse is finished, the OFT instrument is thoroughly wiped with 75% ethanol.

[0054] Experimental principle: Mice prefer to be in the dark, so they prefer to move around the experimental box, but healthy mice are curious and exploratory, which makes them explore the central area, while anxious mice will have less exploratory behavior, so anxious mice will have less activity in the central area of ​​the open experimental box and more stereotyped behavior. The shorter the time the mouse stays in the central area of ​​the reaction box, the higher the anxiety level of the mouse.

[0055] (2) Elevated plus maze (EPM).

[0056] The elevated plus maze consists of open arms and closed arms that cross in a cross shape. The middle area of ​​the intersection is called the central area. The two arms are 10 cm wide and 35 cm long. The closed arms are 15 cm high. The maze is about 40 cm above the ground.

[0057] Experimental principle: The curiosity of mice will prompt them to explore the new open and bright horizon (open arm), but because of their nature of preferring darkness, they are willing to stay in the closed arm. The height of the mouse in the elevated maze is equivalent to that of a human on the edge of a cliff, and the animal is prone to fear and anxiety. The total number of times the mouse enters the open arm and the closed arm, the proportion of each, and the time it stays in the open arm and the closed arm can reflect the anxiety level of the mouse. Experimental steps: a. Take the test mouse out of the cage and gently place it in the central area of ​​the cross maze facing the open arm from the same position and angle. After placing the mouse, the experimenter should leave the behavior room quickly and quietly; b. Record the whereabouts of the mouse through a video tracking system, and count the number of times the mouse enters the open arm (openarm entry, OE), the number of times the mouse enters the closed arm (closed arm entry, CE), the active time in the open arm (openarm time, OT) and the active time in the closed arm (closedarm time, CT), the proportion of the number of times the mouse enters the open arm (OE%), that is, OE / (OE+CE), the proportion of the active time in the open arm (OT%), that is, OT / (OT+CT), and the experimental time for each mouse is 5 minutes; c. After the experiment of each experimental mouse, wipe the maze with a paper towel containing 75% alcohol to remove the odor, urine and feces left by the mouse.

[0058] (3) Y-maze.

[0059] The Y-maze consists of three equal-length closed arms A, B, and C (length 40 cm, width 10 cm, height 16 cm), with an angle of 120 degrees between each two arms, and the maze frame is 40 cm above the ground. The Y-maze is widely used in research fields such as functional impairment, tropism, and spatial working memory.

[0060] Experimental steps: a. On the first day, the mouse was gently placed in the center of the Y maze from the same angle and position. The experimenter quietly left the behavior room and let the mouse freely explore and adapt to the maze environment for 8 minutes. b. The formal experiment was divided into two times. We repeated the above operation one hour after adapting to the two same objects and on the second day. The whereabouts of the mouse were recorded by the video tracking system. The order in which the mouse entered the three arms A, B, and C was counted. The standard for entering the arm was that all four feet of the mouse entered the arm once. Statistics ① The total number of entries m (the total number of entries): the total number of times the mouse entered the three maze arms; ② The number of valid behaviors n (analtermation): entering all three arms of the Y maze in sequence is counted as one valid behavior. ③ Valid behavior score = n / (m-2)*100%. The higher the valid behavior score, the stronger the working memory ability of the mouse.

[0061] (4) New object recognition (NOR).

[0062] The experimental equipment includes: a set of equipment for open field experiment: a square box (42cm long × 42cm wide × 42cm high), a video tracking system and behavioral analysis software; three objects A, B, and C. Objects A and B are exactly the same, and object C is very different from objects A and B, but the specifications and sizes are similar; generally mice need objects with a diameter of about 3cm; it is better for the three objects to be round as much as possible, as mice tend to prefer cubes or rectangular prisms with angles.

[0063] Experimental principle: The experimental mice's natural exploration of new things determines that they are more curious about new objects than familiar old objects. The cognitive memory ability of experimental mice is evaluated by behavioral methods to evaluate the length of time the experimental mice explore familiar old objects and new unfamiliar objects.

[0064] Experimental procedures: The novel object recognition (NOR) experiment is mainly divided into the following three stages, namely the adaptation period, the familiarization period and the test period: a. The first stage - the adaptation period: no objects are placed in the experimental box, and the mouse is placed in the middle of the experimental box for 10 minutes to allow it to adapt to the environment. b. The second stage - the familiarization period: two identical objects (AB) A and B are placed in the experimental box (make sure the objects have no odor and are fixed and cannot be moved), and the objects are 10 cm away from the inner wall of the experimental box. The mouse is placed in the experimental box at an equal distance from the two objects, with its back facing the experimental objects. The experimenter quietly leaves and allows the mouse to explore freely. The video tracking system records the exploration time of the mouse for each object within 5 minutes (effective exploration of the object is considered to be within 2-3 cm of the mouth or nose approaching the object). c. The third stage - the test period: 1 hour after the end of the second stage. Replace the B object in the experimental box with another C object (AC) of different shape and color, make sure that the C object is fixed and cannot be moved, and gently put the mouse in from the same position, and observe and record the time the mouse explores the A and C objects within 5 minutes. After each mouse completed an exploration, the experimental box and the surface of the objects were wiped with a paper towel containing 75% alcohol to remove the smell left by the mouse on the objects.

[0065] 1.6 Immunofluorescence of brain slices.

[0066] (1) Fixation: Remove the whole brain after perfusion, wash the surface blood with 1× PBS, place it in tissue fixative, and fix it at 4°C overnight for more than 24 hours; (2) Dehydration: After fixation, wash away the fixative with 1× PBS and dehydrate with 30% hypertonic sucrose solution; (3) Embedding: Before sectioning, place the tissue in an embedding box, apply OCT embedding medium on the surface, and place it in a -80℃ refrigerator overnight; (4) Frozen sections: Use a cryostat to cut the tissue into pieces with a thickness of about 10 μm. Place the slices directly on a glass slide at room temperature and leave them at room temperature for 1 h or dry them at 37°C for 30 min. When the tissue slices are firmly attached, wash off the surface OCT with 1× PBS. (5) Blocking and permeabilization: Thaw 0.3% Triton X-100-5% BSA-PBS in advance, circle the tissue with an immunohistochemistry pen, draw an appropriate amount and drop it on the slice, place it in a wet box, and block it at room temperature for 1 h; after blocking, wash off the blocking solution with 1× PBS; (6) Primary antibody: Use an equal volume of ddH2O to dilute 2×GDB to 1×GDB, dilute the antibody proportionally, drop it on the surface of the slice, place it in a humidified box, and incubate it at 4°C for 48 h; (7) Secondary antibody: Wash away the primary antibody and select the secondary antibody based on the species of the primary antibody; (8) Nuclear staining: add 5 μl / slice of DAPI to the tissue surface, incubate for 5 min, then wash with 1× PBS and wait until dry; (9) Sealing: Add 5 μl / tissue of tissue sealing agent on the surface of the tissue and cover with a coverslip; leave at room temperature until the sealing agent is basically solidified, and fix it with nail polish.

[0067] 1.7 Construction of AVV-VEGF-C and related experiments (1) Construction of adenovirus shuttle plasmid: ① Overexpression of target gene: The coding gene of vascular endothelial growth factor C (VEGF-C) (CDS sequence of VEGF-C, as shown in SEQ ID No. 1) was constructed on pAdTrack-CMV through appropriate restriction sites, and restriction digestion identification and sequencing of the shuttle plasmid were performed; ② Knockdown of target gene: Design and synthesize DNA fragments encoding shRNA according to the target gene, construct them on pAdTrack-U6 through SalI-BglII, and identify the shuttle plasmid by enzyme digestion and sequencing; ③After sequencing is correct, amplify and purify the DNA in large quantities and store it at -20°C; ④Verification of plasmid validity: Use Western blot or RT-qPCR to quantitatively detect the expression level of the target gene.

[0068] (2) Construction of adenovirus recombinant plasmid: ① Shuttle plasmid linearization: pAdTrack-CMV / U6 was digested with PmeI, and then the linearized plasmid was extracted and recovered using phenol chloroform; ② Transform it into competent bacteria (BJ5183) containing auxiliary plasmid pAdEasy, spread it on LB agar plates containing kanamycin (50-100ug / mL), incubate it at 37℃ overnight, pick 7-8 single clones for shake amplification, extract plasmid by alkaline lysis method, digest it with PacI, and observe whether the recombinant is constructed correctly by 0.7% agarose gel electrophoresis; ③ Extract the recombinant plasmid in large quantities, digest it with PacI, extract it with phenol chloroform, and prepare to transfect 293A cells.

[0069] (3) Recombinant adenovirus packaging: ① Inoculate 1×10 6 293A cells were cultured for 24 h and transfected when the confluence reached 60-70%. ②Use the linearized recombinant plasmid to transfect cells and change the medium the next day; ③ After 7-10 days, transfer the cells to a 50 mL centrifuge tube, resuspend the cells in 2 mL HBSS or PBS after centrifugation; place in a methanol ice bath, freeze and thaw three times at 37°C; collect the supernatant after brief centrifugation and store at -80°C; ④ Virus supernatant infection experiment: Use the above virus supernatant to infect 293A cells; ⑤ Collect the virus as in step ③, and amplify the virus in large quantities according to the above steps.

[0070] (4) Injection of VEGF-C-encoding adenovirus AVV (AVV-VEGF-C) into the cerebellar and medullary cisterna magna: The mouse occipital skin area of ​​0.5 cm × 0.5 cm was prepared and routinely disinfected. The skin surface was anesthetized with 0.1 mL of 2% lidocaine. The skin was cut about 1 cm along the midline of the occipital region with scissors. The subcutaneous and muscle tissues were bluntly separated with forceps. The cerebellomedullary cisterna was freed and exposed. A 34GA needle connected to a Hamilton microsyringe was carefully inserted into the cerebellomedullary cisterna about 2 mm. The needle was injected at a rate of 1 μL / min. Adenovirus AVV-VEGF-C or AVV-control was injected slowly, totaling 3 μL. After the injection, the needle was left in place for 3 minutes to prevent cerebrospinal fluid reflux; finally, the needle was removed, and absorbable biological glue was used to seal the meninges and skin for 4 weeks.

[0071] The information of the gene encoding vascular endothelial growth factor C in the gene database is as follows: >lcl|NM_009506.2_cds_NP_033532.1_1 [gene=Vegfc] [db_xref=CCDS:CCDS22306.1][protein=vascular endothelial growth factor C precursor][protein_id=NP_033532.1][location=248..1495] [gbkey=CDS]; The coding gene sequence of vascular endothelial growth factor C is shown in SEQ ID No. 1:

[0072] 2. Experimental results.

[0073] 2.1 Septic mice have impaired cerebral glymphatic system reflux.

[0074] The present invention injects the magnetic resonance imaging (MRI) paramagnetic contrast agent Gd-DOTA (simulating metabolic waste in the brain) into the cerebellomedullary cistern of sham and cecal ligation and puncture (CLP) sepsis mouse models. The specific method is that 24 hours after the CLP sepsis model mice are modeled, the cerebellomedullary cistern of the mice is surgically exposed, and the paramagnetic contrast agent Gd-DOTA is injected to simulate metabolic waste in the brain. After Gd-DOTA circulates in the cerebrospinal fluid for a period of time, the mice are transferred to a 9.4T magnetic resonance for data scanning and acquisition. Finally, the longitudinal relaxation time quantitative imaging (T1 mapping) technology newly developed based on the MRI VFA-SPGR sequence is used to simulate and calculate the mouse brain T1 map using six continuous small-angle gradient echo sequences. Its imaging has sufficient time and spatial resolution, and can record the excretion pathway of Gd-DOTA from the brain to the cervical lymph nodes in real time, quantitatively and dynamically in living animals. The present invention is innovatively applied to the imaging of the glial lymphatic system in the brain of septic mice, objectively and accurately describing the brain metabolic waste transport function in the pathophysiological process of sepsis, and visualizing the key physiological, physical processes and fine anatomical structures of cerebrospinal fluid transport and entry and exit of brain parenchyma. Through scanning, processing, simulation and analysis, the T1 map of the mouse brain was calculated by MRI simulation of 6 different small-angle spoiled gradient echo sequences (see the method section for the specific calculation method). Longitudinal relaxation time quantitative imaging and voxel-based morphological analysis found that quantitative analysis of brain regions showed that compared with the Sham group mice, the T1 values ​​of various brain regions (hippocampus, cerebellum, hypothalamus, olfactory bulb, and thalamus) of the CLP group mice were significantly increased, indicating that the clearance efficiency of Gd-DOTA injected into the cerebrospinal fluid in septic mice was significantly reduced, and there was a serious brain glial lymphatic system transport disorder ( Figure 1 , Figure 2 ).

[0075] The transport of cerebrospinal fluid by the glymphatic system mainly depends on the aquaporin 4 (AQP4) on the "endfeet" of the vascular side of astrocytes. Through the paravascular and "endfeet" pathways, it completes the exchange of cerebrospinal fluid (CSF) and interstitial fluid (ISF), removes metabolic waste from the brain, maintains the stability of extracellular ions and fluids, and provides the best internal environment for normal neural activity in the brain. AQP4 protein is mainly distributed on the plasma membrane of the "endfeet" of astrocytes. This phenomenon is called AQP4 polarity. Abnormal expression of AQP4 protein or loss of polarity can weaken the function of the glymphatic system. In addition, upregulated expression of AQP4 and excessive activation of astrocytes indicate that individuals with sepsis have severe neuroinflammatory reactions, that is, septic encephalopathy. Using GFAP to label astrocytes, brain slice immunofluorescence results showed that the AQP4 / GFAP fluorescence intensity in sepsis model mice in the CLP group was upregulated ( Figure 3 Figure A and Figure B). Correlation analysis showed that the fluorescence intensity of AQP4 / GFAP (the degree of neuroinflammation) in the sepsis model mice in the CLP group was positively correlated with the T1 value of the corresponding mouse MRI longitudinal relaxation time quantitative imaging, indicating that the degree of neuroinflammation in the sepsis model mice in the CLP group was significantly correlated with the transport disorder of the brain glial lymphatic system, that is, the worse the clearance function of the glial lymphatic system, the more severe the neuroinflammation of the septic mice, and the higher the probability and severity of their concurrent septic encephalopathy ( Figure 3 Figure C).

[0076] 2.2 Impaired glymphatic system reflux in septic mice can predict cognitive dysfunction.

[0077] Cognitive dysfunction caused by brain damage caused by sepsis is becoming increasingly prominent in clinical practice, manifested as decreased memory function and anxiety, which reduces the quality of life of patients. Therefore, it is of positive significance to pay attention to the prevention and treatment of septic encephalopathy and cognitive dysfunction caused by sepsis. The present invention uses open field test and cross elevated test to detect anxiety-like behavior in cognitive dysfunction of CLP model septic mice, and uses Y maze test and novel object recognition test to detect memory impairment behavior in cognitive dysfunction of CLP model septic mice. The specific methods are shown in the methodology section. The results showed that the time that mice in the CLP group stayed in the central area of ​​the open field was significantly reduced, indicating that they had anxiety-like behavior. Correlation analysis showed that the time they stayed in the central area of ​​the open field was negatively correlated with the T1 value of the corresponding mouse MRI longitudinal relaxation time quantitative imaging, that is, the worse the clearance function of the glial lymphatic system, the more severe the anxiety-like emotion of the septic mice, and the higher the probability and severity of their concurrent septic encephalopathy ( Figure 4). The results of the novel object recognition experiment showed that the CLP group mice had significantly reduced ability to recognize novel objects, suggesting that they had impaired memory. Correlation analysis showed that the exploration time of novel objects was negatively correlated with the T1 value of the corresponding mouse MRI longitudinal relaxation time quantitative imaging, that is, the worse the clearance function of the glial lymphatic system in septic mice, the more severely impaired their memory function was, and the higher the probability and severity of their concurrent septic encephalopathy was ( Figure 5 The results of the Y-maze experiment showed that the CLP group mice had a significantly reduced novel arm rotation ratio (effective number of explorations) in the Y-maze experiment, suggesting that they had impaired memory. Correlation analysis showed that the novel arm rotation ratio was negatively correlated with the T1 value of the corresponding mouse MRI longitudinal relaxation time quantitative imaging, that is, the worse the clearance function of the glial lymphatic system, the more severe the memory impairment of the septic mice, and the higher the probability and severity of their concurrent septic encephalopathy ( Figure 6 ). In the cross-elevation experiment, the CLP group mice spent significantly less time in the open arms, suggesting that they had anxiety-like behaviors. Correlation analysis showed that the time they spent in the open arms was negatively correlated with the T1 value of the corresponding MRI longitudinal relaxation time quantitative imaging of the mice, that is, the worse the clearance function of the glial glymphatic system, the worse the memory function of the septic mice, and the higher the probability and severity of their concurrent septic encephalopathy ( Figure 7 ).

[0078] 2.3 The glial lymphatic system reflux disorder in sepsis model mice is associated with the activation of type A1 astrocytes.

[0079] In 2017, Liddelow's team first reported in Nature that LPS and middle cerebral artery occlusion induced two different types of reactive astrocytes, called type A1 (neurotoxic) and type A2 (neuroprotective). C3 is a marker for type A1 reactive astrocytes, and its upregulation indicates excessive neurotoxic astrocyte activation and neuroinflammation in the brain. GFAP was used to label astrocytes, and C3 was used to label type A1 activated astrocytes. The results of brain slice immunofluorescence showed that the C3 / GFAP fluorescence intensity was upregulated in the sepsis model mice in the CLP group ( Figure 8 A and B in the middle). Correlation analysis showed that the C3 / GFAP fluorescence intensity (activation degree of A1 type astrocytes) of the sepsis model mice in the CLP group was positively correlated with the T1 value of the corresponding mouse MRI longitudinal relaxation time quantitative imaging, indicating that the activation degree of A1 type astrocytes in the sepsis model mice in the CLP group was significantly correlated with the transport disorder of the brain glial lymphatic system, that is, the worse the clearance function of the glial lymphatic system, the more severe the neurotoxic astrocyte activation and neuroinflammation of the septic mice, and the higher the probability and severity of their concurrent septic encephalopathy ( Figure 8 Middle C).

[0080] 2.4 Promoting and improving the return of the glymphatic system is a potential therapeutic target for septic encephalopathy.

[0081] Vascular endothelial growth factor C (VEGF-C) is a lymphatic vessel-specific growth factor that can promote the proliferation and migration of lymphatic endothelial cells and induce the differentiation of peripheral blood endothelial progenitor cells into lymphatic endothelial cells, thereby promoting the formation of lymphatic vessels. It is a classic way to improve and promote the return of brain glial lymphatic system. The adenovirus AVV encoding VEGF-C (AVV-VEGF-C, Fig. 9 Figure A proves that the construction was successful) was injected into the cisterna magna of the mouse cerebellum and medulla oblongata. Four weeks later, the contrast agent Gd-DOTA was injected for 9.4T magnetic resonance scanning to obtain the brain and neck T1 maps. The distribution, uptake and transport patterns of Gd-DOTA between the AVV-VEGF-C and negative control virus AVV-Control groups were compared through ROIs analysis of various brain regions, cluster analysis of T1 values ​​in the nasal concha region and quantitative analysis of T1 in deep cervical lymph nodes. Behavioral tests were performed to detect the cognitive function of mice after CLP modeling.

[0082] The experimental results showed that adenovirus AAV-VEGF-C can not only stimulate the development and upregulation of meningeal lymphatic vessels, but also stimulate the proliferation of deep cervical lymph nodes and enhance the cerebrospinal fluid transport and drainage efficiency of the glial lymphatic system ( Fig. 9 ). Behavioral studies suggest that mice have severe cognitive dysfunction after CLP modeling: the open field experiment showed that compared with the Sham+AVV-Control control group, the activity distance of mice in the CLP+AVV-Control group in the central area of ​​the open field was significantly reduced, suggesting that they have anxiety-like behavior. Correlation analysis showed that their anxiety-like degree was significantly correlated with the transport disorder of the brain gliolymphatic system ( Fig.10 ); The Y-maze experiment showed that compared with the Sham+AVV-Control control group, the percentage of alternations in the novel arm of the CLP+AVV-Control group mice was significantly reduced, indicating that they had severe memory impairment. Correlation analysis showed that the degree of memory impairment was significantly correlated with the transport disorder of the brain gliolymphatic system ( Fig.11 ); The novel object recognition experiment showed that compared with the Sham + AVV-Control control group, the CLP + AVV-Control group mice had significantly less interest in exploring new objects, suggesting that they had severe memory impairment. Correlation analysis showed that the degree of memory impairment was significantly correlated with the brain gliolymphatic system transport disorder ( Fig.12); The cross-elevation test showed that compared with the Sham+AVV-Control control group, the percentage of time that the mice in the CLP+AVV-Control group entered the open arm was significantly reduced, indicating that they had significant anxiety-like behavior. Correlation analysis showed that their anxiety-like degree was significantly correlated with the transport disorder of the brain gliolymphatic system ( Fig.13 ). Injection of adenovirus AAV-mVEGF-C, which promotes the return of the glymphatic system, can significantly reverse this cognitive impairment, indicating that enhancing the function of meningeal lymphatic vessels and promoting the return of the glymphatic system may be a promising therapeutic target for the treatment of septic encephalopathy.

[0083] In summary, the present invention discovered for the first time that the glial lymphatic system can effectively diagnose whether sepsis model mice have septic encephalopathy, as well as the severity of cognitive dysfunction, neuroinflammation, and astrocyte A1 activation. Promoting and improving the reflux of the glial lymphatic system is an effective therapeutic target for septic encephalopathy. The present invention provides a new idea for the preparation of diagnostic and therapeutic products for septic encephalopathy, and has broad application prospects.

Claims

1. Use of a reagent for detecting brain glial lymphatic system reflux disorder in the preparation of a product for diagnosing septic encephalopathy, characterized in that: The reagent for detecting brain gliolymphatic system reflux disorder includes magnetic resonance paramagnetic contrast agent Gd-DOTA for brain medullary cisterna magna injection.

2. The use of the reagent for detecting brain glial lymphatic system reflux disorder according to claim 1 in the preparation of a product for diagnosing septic encephalopathy, characterized in that: The septic encephalopathy is septic encephalopathy simulated by a cecal ligation and puncture model.

3. Use of a product for promoting the reflux of the glial lymphatic system in the preparation of a product for treating septic encephalopathy, characterized in that: The product promoting the reflux of the glial lymphatic system is a gene expression product capable of encoding and synthesizing vascular endothelial growth factor C. The gene encoding the vascular endothelial growth factor C is shown in SEQ ID No.

1.

4. The use of the product for promoting the reflux of the glial lymphatic system according to claim 3 in the preparation of a product for treating septic encephalopathy, characterized in that: The septic encephalopathy is septic encephalopathy simulated by a cecal ligation and puncture model.

5. The use of the product for promoting the reflux of the glial lymphatic system according to claim 3 in the preparation of a product for treating septic encephalopathy, characterized in that: The expression vector of the gene expression product is adenovirus.

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