Vascular Endothelial Growth Factor-C Treatment Enhances Cerebrospinal Fluid Outflow during Toxoplasma gondii Brain Infection but Does Not Improve Cerebral Edema
2023; Elsevier BV; Volume: 194; Issue: 2 Linguagem: Inglês
10.1016/j.ajpath.2023.11.008
ISSN1525-2191
AutoresMichael A. Kovacs, Isaac W. Babcock, Ana Royo Marco, Lydia A. Sibley, Abigail G. Kelly, Tajie H. Harris,
Tópico(s)Spinal Dysraphism and Malformations
ResumoCerebral edema frequently develops in the setting of brain infection and can contribute to elevated intracranial pressure, a medical emergency. How excess fluid is cleared from the brain is not well understood. Previous studies have shown that interstitial fluid is transported out of the brain along perivascular channels that collect into the cerebrospinal fluid (CSF)–filled subarachnoid space. CSF is then removed from the central nervous system through venous and lymphatic routes. The current study tested the hypothesis that increasing lymphatic drainage of CSF would promote clearance of cerebral edema fluid during infection with the neurotropic parasite Toxoplasma gondii. Fluorescent microscopy and magnetic resonance imaging was used to show that C57BL/6 mice develop vasogenic edema 4 to 5 weeks after infection with T. gondii. Tracer experiments were used to evaluate how brain infection affects meningeal lymphatic function, which demonstrated a decreased rate in CSF outflow in T. gondii–infected mice. Next, mice were treated with a vascular endothelial growth factor (VEGF)-C–expressing viral vector, which induced meningeal lymphangiogenesis and improved CSF outflow in chronically infected mice. No difference in cerebral edema was observed between mice that received VEGF-C and those that rececived sham treatment. Therefore, although VEGF-C treatment can improve lymphatic outflow in mice infected with T. gondii, this effect does not lead to increased clearance of edema fluid from the brains of these mice. Cerebral edema frequently develops in the setting of brain infection and can contribute to elevated intracranial pressure, a medical emergency. How excess fluid is cleared from the brain is not well understood. Previous studies have shown that interstitial fluid is transported out of the brain along perivascular channels that collect into the cerebrospinal fluid (CSF)–filled subarachnoid space. CSF is then removed from the central nervous system through venous and lymphatic routes. The current study tested the hypothesis that increasing lymphatic drainage of CSF would promote clearance of cerebral edema fluid during infection with the neurotropic parasite Toxoplasma gondii. Fluorescent microscopy and magnetic resonance imaging was used to show that C57BL/6 mice develop vasogenic edema 4 to 5 weeks after infection with T. gondii. Tracer experiments were used to evaluate how brain infection affects meningeal lymphatic function, which demonstrated a decreased rate in CSF outflow in T. gondii–infected mice. Next, mice were treated with a vascular endothelial growth factor (VEGF)-C–expressing viral vector, which induced meningeal lymphangiogenesis and improved CSF outflow in chronically infected mice. No difference in cerebral edema was observed between mice that received VEGF-C and those that rececived sham treatment. Therefore, although VEGF-C treatment can improve lymphatic outflow in mice infected with T. gondii, this effect does not lead to increased clearance of edema fluid from the brains of these mice. Cerebral edema is a common manifestation of brain infection that results from excess fluid accumulation in the brain parenchyma. Because the skull is a rigid compartment, swelling of the brain can lead to elevated intracranial pressure, a pathogenic state that must be closely monitored because of the potential for ischemic injury and life-threatening brain herniation.1Koenig M.A. Cerebral edema and elevated intracranial pressure.Continuum (Minneap Minn). 2018; 24: 1588-1602Google Scholar Current therapies for cerebral edema are limited and include corticosteroids, osmotic agents, and more invasive surgical interventions, such as decompressive craniectomy.2Cook A.M. Morgan Jones G. Hawryluk G.W.J. Mailloux P. McLaughlin D. Papangelou A. Samuel S. Tokumaru S. Venkatasubramanian C. Zacko C. Zimmermann L.L. Hirsch K. Shutter L. 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Lymphatic endothelium: morphological, molecular and functional properties.J Cell Biol. 2003; 163: 209-213Google Scholar Failure of lymphatic drainage can be a complication of lymph node resection or infection and can lead to intractable tissue swelling in the extremities (known as lymphedema).4Witte M.H. Bernas M.J. Martin C.P. Witte C.L. Lymphangiogenesis and lymphangiodysplasia: from molecular to clinical lymphology.Microsc Res Tech. 2001; 55: 122-145Google Scholar,5Alitalo K. Tammela T. Petrova T.V. Lymphangiogenesis in development and human disease.Nature. 2005; 438: 946-953Google Scholar Fluid clearance from the brain is more complicated because conventional lymphatic vessels do not develop within the tissue parenchyma. Instead, interstitial fluid diffuses into nearby perivascular spaces and travels by bulk flow along penetrating arteries or veins into the cerebrospinal fluid (CSF)–filled subarachnoid space.6Hladky S.B. 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Evidence supporting a role for lymphatic drainage in this process comes from experimental rodent studies in which surgically impairing cervical lymphatic outflow worsened cerebral edema after middle cerebral artery occlusion–induced ischemic stroke or subarachnoid hemorrhage.15Sun B.L. Xia Z.L. Yan Z.W. Chen Y.S. Yang M.F. Effects of blockade of cerebral lymphatic drainage on cerebral ischemia after middle cerebral artery occlusion in rats.Clin Hemorheol Microcirc. 2000; 23: 321-325Google Scholar, 16Si J. Chen L. Xia Z. Effects of cervical-lymphatic blockade on brain edema and infarction volume in cerebral ischemic rats.Chin J Physiol. 2006; 49: 258-265Google Scholar, 17Sun B.L. Xia Z.L. Wang J.R. Yuan H. Li W.X. Chen Y.S. Yang M.F. Zhang S.M. 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Shi Z. Lu H. Wang L. Fan W. Tong X. Yan H. Improving the function of meningeal lymphatic vessels to promote brain edema absorption after traumatic brain injury.J Neurotrauma. 2022; 40: 383-394Google Scholar VEGF-C therapy has begun to show some success in human studies for the treatment of secondary lymphedema.24Leppapuska I.M. Hartiala P. Suominen S. Suominen E. Kaartinen I. Maki M. Seppanen M. Kiiski J. Viitanen T. Lahdenpera O. Vuolanto A. Alitalo K. Saarikko A.M. Phase 1 lymfactin(R) study: 24-month efficacy and safety results of combined adenoviral VEGF-C and lymph node transfer treatment for upper extremity lymphedema.J Plast Reconstr Aesthet Surg. 2022; 75: 3938-3945Google Scholar Therefore, the beneficial effect of VEGF-C treatment on cerebral edema in experimental animal models may have the potential for clinical translation. This study examined the contribution of meningeal lymphatic drainage to the control of cerebral edema in a murine model of chronic brain infection. To investigate this process, the model organism Toxoplasma gondii, a neurotropic parasite, was used that establishes a well-controlled latent infection in the brains of mice characterized by periodically reactivating cysts.25Di Cristina M. Marocco D. Galizi R. Proietti C. Spaccapelo R. Crisanti A. Temporal and spatial distribution of Toxoplasma gondii differentiation into bradyzoites and tissue cyst formation in vivo.Infect Immun. 2008; 76: 3491-3501Google Scholar,26Ferguson D.J. Hutchison W.M. Pettersen E. Tissue cyst rupture in mice chronically infected with Toxoplasma gondii: an immunocytochemical and ultrastructural study.Parasitol Res. 1989; 75: 599-603Google Scholar Significant vasogenic edema was observed in infected regions of the brain, confirmed by Evans blue permeability assays and quantitative measurement of brain water content. CSF tracer studies revealed that meningeal lymphatic drainage was impaired during the chronic stage of T. gondii infection, similar to that in studies using a model of viral encephalitis.27Li X. Qi L. Yang D. Hao S. Zhang F. Zhu X. Sun Y. Chen C. Ye J. Yang J. Zhao L. Altmann D.M. Cao S. Wang H. Wei B. Meningeal lymphatic vessels mediate neurotropic viral drainage from the central nervous system.Nat Neurosci. 2022; 25: 577-587Google Scholar Adeno-associated virus (AAV)–mediated delivery of VEGF-C induced lymphatic vessel growth in the meninges and led to a recovery in CSF outflow to the deep cervical lymph nodes. However, this improvement did not have any beneficial effect on water clearance from the brain. Thus, these results suggest that VEGF-C–mediated expansion of lymphatic vessels in the meninges does not enhance removal of cerebral edema fluid during T. gondii brain infection. All mice were housed at University of Virginia (Charlottesville, VA) pathogen-free facilities with a 12-hour light/dark cycle. C57BL/6J (number 000664) and CBA/J (number 000656) strains were originally purchased from the Jackson Laboratory (Bar Harbor, ME) and then maintained within the authors' animal facility. Swiss Webster (number 024) mice were purchased from Charles River Laboratories (Wilmington, MA) and maintained within the authors' animal facility. For experiments reported in this study, age-matched young adult female mice were used. Animals were infected at 7 to 9 weeks of age, and AAV injections were performed at 5 weeks of age. All experiments were approved by the Institutional Animal Care and Use Committee at the University of Virginia under protocol number 3968. The avirulent, type II ME49 strain of T. gondii was used for all infections. The parasite was maintained in chronically infected (2 to 6 months) Swiss Webster mice and passaged through CBA/J mice. For infections, tissue cysts were prepared from homogenized brains of chronically infected (4 to 8 weeks) CBA/J mice. Experimental mice were then inoculated intraperitoneally with 10 tissue cysts of ME49 in 200 μL of 1× phosphate-buffered saline (PBS). Whole brains were weighed immediately after harvest for measurement of the wet weight. Brains were then dehydrated in an oven at 96°C for 24 hours to obtain the dry weight. Percentage water content was calculated as follows: (wet weight − dry weight)/(wet weight) × 100. To assess BBB permeability, i.v. injection of 2% Evans blue dye (Sigma-Aldrich, St. Louis, MO) diluted in 200 μL of 1× PBS was performed using the retro-orbital approach. At 1 hour after dye administration, mice were sacrificed and transcardiac perfusion was performed using 20 mL of cold 1× PBS. For confocal microscopy experiments, see Confocal Microscopy. For quantitative measurement of Evans blue tissue extravasation, whole brains were harvested and bisected along the sagittal midline. One hemisphere was immersed in 2 mL of formamide (Sigma-Aldrich) at 37°C overnight to extract the Evans blue dye. Extracted dye was then quantified by spectrophotometry using an optical absorbance of 620 nm and compared to a standard curve. For analysis of Evans blue dye extravasation into the brain parenchyma, transcardiac perfusion of Evans blue dye–injected mice was performed using 1× PBS, followed immediately after by transcardiac perfusion with cold 4% paraformaldehyde (PFA). Whole brains were then harvested, bisected along the sagittal midline, and post-fixed in cold 4% PFA for 24 hours. Brains were cryoprotected in 30% sucrose for 24 hours at 4°C, embedded in Tissue Tek OCT (Sakura Finetek, Torrance, CA), and frozen on dry ice. The sections (50 μm thick) were cut using a CM 1950 cryostat (Leica Biosystems, Deer Park, IL) and stored in cold 1× PBS. To immunostain free-floating sections, tissue sections were incubated in a blocking solution (2% normal donkey serum, 1% bovine serum albumin, 0.05% Tween 20, and 0.5% Triton X-100 in 1× PBS) at room temperature for 45 minutes. Sections were then incubated with primary antibody overnight at 4°C (rabbit polyclonal α-ME49 at 1:10,000 dilution; a gift from Fausto Araujo, Palo Alto Medical Foundation, Mountain View, CA). Samples were washed three times in a solution of 0.05% Tween 20 (in 1× PBS) and incubated with secondary antibody (donkey α-rabbit-AF488 at 1:500 dilution; Thermo Fisher Scientific, Waltham, MA) for 1 hour at room temperature. Finally, tissues were counter-stained with DAPI (Thermo Fisher Scientific), washed three times in a solution of 0.05% Tween 20 (in 1× PBS), mounted onto glass slides using AquaMount (Thermo Fisher Scientific), and coverslipped. Images were acquired using a Leica TCS SP8 confocal microscope, with fluorescence emission of Evans blue dye detected in the far-red channel (peak emission at 680 nm). For analysis of drainage of intracisternally injected OVA-AF594 to the deep cervical lymph nodes (DCLNs), the DCLNs were harvested bilaterally 1 hour after injection and fixed overnight in 4% PFA at 4°C. The next day, fixed DCLNs were cryopreserved in 30% sucrose for at least 4 hours at 4°C, embedded in OCT (Tissue Tek), and frozen on dry ice. The sections (30 μm thick) were cut using a CM 1950 cryostat (Leica) and mounted onto frosted glass slides (Globe Scientific, Mahwah, NJ). Tissue sections were washed three times in a solution of 0.05% Tween 20 (in 1× PBS), counterstained using DAPI (Thermo Fisher Scientific), and coverslipped using AquaMount. Images were acquired using a Leica TCS SP8 confocal microscope. Quantitative analysis of OVA-AF594 drainage was performed using Fiji software (https://fiji.sc).28Schindelin J. Arganda-Carreras I. Frise E. Kaynig V. Longair M. Pietzsch T. Preibisch S. Rueden C. Saalfeld S. Schmid B. Tinevez J.Y. White D.J. Hartenstein V. Eliceiri K. Tomancak P. Cardona A. Fiji: an open-source platform for biological-image analysis.Nat Methods. 2012; 9: 676-682Google Scholar Three representative sections for each lymph node sample per animal were imaged, and area coverage was calculated as follows: (area of OVA-AF594 signal/area of DAPI signal) × 100. For analysis of lymphatic vessel growth in the dura mater layer of meninges, the dorsal aspect of the skull was harvested and the dural meninges were fixed, while still attached to the skull bone, in 4% PFA for 6 to 8 hours at 4°C. Then, as previously described,29Louveau A. Filiano A.J. Kipnis J. Meningeal whole mount preparation and characterization of neural cells by flow cytometry.Curr Protoc Immunol. 2018; 121: e50Google Scholar whole mount dural meninges were carefully dissected and stored in 1× PBS. To immunostain whole mount meninges, the fixed tissues were first incubated in a blocking solution (as described earlier in this paragraph) at room temperature for 45 minutes. Then, tissue was stained for 2 hours at room temperature with directly conjugated primary antibody [lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1)-EF660 at 1:200; eBioscience, San Diego, CA; catalog number 50-0443-82]. Tissue was counter-stained with DAPI (Thermo Fisher Scientific), washed three times in a solution of 0.05% Tween 20 (in 1× PBS), mounted onto glass slides using AquaMount, and coverslipped. Images were acquired using a Leica TCS SP8 confocal microscope. Quantitative analysis of lymphatic vessel growth was performed using Fiji software. Area coverage was calculated for each sample in a field of view centered on the confluence of sinuses: (area of LYVE1 signal/area of field of view) × 100. Mice were anesthetized by i.p. injection of a solution containing ketamine (100 mg/kg) and xylazine (10 mg/kg) diluted in saline. Mice were secured in a stereotaxic frame with the head angled slightly downward. An incision in the skin was made at the base of the skull, and the muscle layers overlying the atlanto-occipital membrane were retracted. A 33-gauge Hamilton (Reno, NV) syringe (number 80308) was inserted at a steep angle through the membrane to inject the desired solution into the CSF-filled cisterna magna. After injection, the skin was closed using 5-0 nylon sutures, and mice received an s.c. injection of ketoprofen (2 mg/kg). Mice were then allowed to recover on a heating pad until awake. To assess meningeal lymphatic function, 3 μL of Alexa Fluor 594–conjugated ovalbumin (OVA-AF594; Thermo Fisher) diluted in artificial CSF (2 mg/mL) was injected into the cisterna magna of mice. At 1 hour after injection, mice were sacrificed, and OVA-AF594 drainage to the DCLNs was measured by confocal microscopy (see Confocal Microscopy) or fluorescence spectroscopy. The latter approach involved DCLN harvest and enzymatic digestion with Collagenase D (1 mg/mL; Sigma-Aldrich) at 37°C for 30 minutes, followed by a 2-hour treatment in T-PER Tissue Protein Extraction Reagent (Thermo Fisher Scientific) at 4°C. Samples were then centrifuged at >12,000 × g to remove cellular debris, and the supernatant containing extracted protein was transferred to a black flat-bottom microwell plate (Millipore Sigma, St. Louis, MO; M9685). Relative fluorescence intensity of each sample was measured on a SpectraMax iD3 microplate reader (Molecular Devices, San Jose, CA) using an excitation wavelength of 590 nm and an emission wavelength of 630 nm and then compared with a standard curve to determine the total amount of CSF tracer that drained. Induction of meningeal lymphangiogenesis was performed as previously described.30Song E. Mao T. Dong H. Boisserand L.S.B. Antila S. Bosenberg M. Alitalo K. Thomas J.L. Iwasaki A. VEGF-C-driven lymphatic drainage enables immunosurveillance of brain tumours.Nature. 2020; 577: 689-694Google Scholar Briefly, 5 μL of AAV1-CMV-VEGFC (Vector BioLabs, Malvern, PA; catalog number AAV-275994) or AAV1-CMV-EGFP (Vector BioLabs; catalog number 7002) was injected directly into the cisterna magna of 5-week–old mice at a concentration of 1 × 1013 genome copies/mL diluted in artificial CSF. Transcardiac perfusion of mice was performed using 20 mL of cold 1× PBS. DCLNs were harvested bilaterally into cold complete RPMI medium [10% fetal bovine serum (Gibco, Grand Island, NY), 1% penicillin/streptomycin (Gibco), 1% sodium pyruvate (Gibco), 1% nonessential amino acids (Gibco), and 0.1% 2-mercaptoethanol (Life Technologies, Carlsbad, CA)], then mechanically homogenized and gently pressed through a 70-μm strainer (Corning Inc., Corning, NY). Cells were pelleted, resuspended, and kept on ice. Brains were harvested into cold complete RPMI medium, minced with a razor blade, and passed through an 18- and a 22-gauge needle for mechanical homogenization. Tissue was then digested in a solution containing collagenase/dispase (0.227 mg/mL; Sigma-Aldrich) and DNase (50 U/mL; Roche, Indianapolis, IN) at 37°C for 45 to 60 minutes, before being passed through a 70-μm strainer (Corning) and washed with complete RPMI medium. Myelin was separated from mononuclear cells by resuspending samples in 20 mL of 40% Percoll (Cytiva, Marlborough, MA) and centrifuging at 650 × g for 25 minutes. Myelin was aspirated, and cell pellets were washed, resuspended, and kept on ice. Cells were treated with 50 μL of Fc block [0.1% rat γ globulin (Jackson ImmunoResearch, West Grove, PA) and 1 μg/mL of 2.4G2 (BioXCell, Lebanon, NH)] for 10 minutes at room temperature. Cells were incubated with phycoerythrin-conjugated I-Ab-AS15 tetramer (NIH Tetramer Core Facility, Emory University, Atlanta, GA) for 15 minutes at room temperature. Cells were then stained for surface markers and incubated with a fixable live/dead viability dye for 30 minutes at 4°C. After surface staining, cells were treated with a fixation/permeabilization solution (eBioscience; 00-5123-43 and 00-5223-56) overnight and then stained for intracellular markers in permeabilization buffer (eBioscience; 00-8333-56) for 30 minutes at 4°C. Finally, samples were resuspended in fluorescence-activated cell sorting buffer (0.2% bovine serum albumin and 2 mmol/L EDTA in 1× PBS) and acquired using a Gallios flow cytometer (Beckman Coulter, Brea, CA). Samples were analyzed using FlowJo software version 10 (Becton Dickinson, Ashland, OR). Cells were stained using the following eBioscience antibodies at 1:200 dilution: TCR-β-APC (17-5961-82), CD4-EF450 (48-0042-82), CD8α-PerCP-Cy5.5 (45-0081-82), and Ki-67-PE-Cy7 (25-5698-82; intracellular). Fixable Viability Dye eFluor 506 (65-0866-18) was used at 1:800 dilution. For assessment of parasite cyst burden, whole brains were minced with a razor blade and passed through an 18- and a 22-gauge needle to mechanically homogenize the tissue. A total of 30 μL of tissue homogenate was mounted onto a microscope slide, and T. gondii cysts were counted using a DM 2000 LED brightfield microscope (Leica). For measurement of parasite burden by quantitative real-time PCR, genomic DNA was isolated from mouse brains, dural meninges, or lymph nodes using the Isolate II Genomic DNA Kit (Bioline, Memphis, TN). Before tissue lysis, whole brains required mechanical homogenization with an Omni TH tissue homogenizer (Omni International, Kennesaw, GA). Quantitative real-time PCR was performed using the CFX384 Real-Time System (Bio-Rad, Hercules, CA) using 500 ng or 1 μg of DNA per reaction, SensiFAST Probe No-ROX Kit (Bioline), and primers specific for a 529-bp repeat region in the T. gondii genome, as previously described.31Cowan M.N. Sethi I. Harris T.H. Microglia in CNS infections: insights from Toxoplasma gondii and other pathogens.Trends Parasitol. 2022; 38: 217-229Google Scholar The number of parasite genomes per sample was quantified using a standard curve generated from purified T. gondii DNA. After an initial 2.0% to 3.0% isoflurane induction period, all mice were anesthetized and maintained with 1.0% to 2.0% isoflurane in oxygen air delivered via a cylindrical nose piece. Body temperature was recorded and maintained at 36°C to 37.5°C using a water-heated stand. Respiratory rate and body temperature were monitored continuously. All MRI imaging was conducted on an actively shielded 9.4-T Bruker Biospec magnet with a 20-cm horizontal bore (Bruker-Biospin, Billerica, MA). An 86-mm inner diameter quadrature volume coil was used as the radiofrequency transmitter, and a four-channel phased array coil was used as the receiver. Scout images in three orthogonal planes were acquired to position the subsequent scans. Local magnetic field homogeneity was optimized within an ellipsoid covering most of the brain using previously acquired field maps. For structural imaging of brain ventricles, a three-dimensional T1-weighted spin-modified driven equilibrium Fourier transform sequence was used with the following parameters: field of view, 20 × 20 × 8 mm; matrix size, 128 × 128 × 30; repetition time, 2635 milliseconds; inversion time, 1000 milliseconds; segment size, 64; one signal average; and a total imaging time of 3 minutes. For lesion observations, a two-dimensional T2-weighted rapid acquisition with refocused echoes sequence was acquired with the following parameters: repetition time, 2500 milliseconds; echo time, 34 milliseconds; field of view, 20 × 20 mm; slice thickness, 0.5 mm; acceleration factor, 8; and four signal averages in 4.5 minutes. Lateral ventricle quantification was done with T1 images imported into HOROS software version 4.0 (https://horosproject.org). Using the two-dimensional viewer, regions of interest were drawn around the lateral ventricles for each imaging plane. Region of interest volume computation was then run within the region of interest suite. Lesions were visualized using FIJI software version 1.54f 29. Statistical analysis was performed using Prism software version 8.4 (GraphPad Software, Boston, MA) or RStudio version 1.1 (https://www.r-project.org) statistical packages. A one-way analysis of variance was performed to compare three or more experimental groups with a single independent variable. When data were combined from multiple experiments, a randomized block analysis of variance was performed using the lme4 software package in R.32Bates D. Mächler M. Bolker B. Walker S. Fitting linear mixed-effects models using lme4.J Stat Software. 2015; 67: 1-48Google Scholar This statistical test models experimental groups as a fixed effect and experimental day as a random effect. The statistical test used for each experiment is defined in the figure legend, and P values are indicated, with NS = not significant, ∗P < 0.05, and ∗∗∗P < 0.001. Graphs were generated using Prism software and show means ± SEM along with individual data points representative of individual mice (biological replicates). Outliers were identified using the ROUT method with a maximum false discovery rate (Q) of 1%.33Motulsky H.J. Brown R.E. Detecting out
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