- Email: [email protected]
Changes in endothelial cell proliferation and vascular permeability after systemic lipopolysaccharide administration in the subfornical organ
Journal of Neuroimmunology 298 (2016) 132–137
Contents lists available at ScienceDirect
Journal of Neuroimmunology journal homepage: www.elsevier.com/locate/jneuroim
Changes in endothelial cell proliferation and vascular permeability after systemic lipopolysaccharide administration in the subfornical organ Shoko Morita-Takemura a,⁎, Kazuki Nakahara a, Kouko Tatsumi a, Hiroaki Okuda a,b, Tatsuhide Tanaka a, Ayami Isonishi a, Akio Wanaka a a b
Department of Anatomy & Neuroscience, Faculty of Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521, Japan Department of Functional Anatomy, Graduate School of Medical Science, Kanazawa University, Kanazawa, Japan
a r t i c l e
i n f o
Article history: Received 5 April 2016 Received in revised form 13 June 2016 Accepted 28 June 2016 Keywords: Angiogenesis Blood–brain barrier Fenestrated endothelial cells Inflammation
a b s t r a c t The subfornical organ (SFO) has highly permeable fenestrated vasculature and is a key site for immune-to-brain communications. Recently, we showed the occurrence of continuous angiogenesis in the SFO. In the present study, we found that systemic administration of bacterial lipopolysaccharide (LPS) reduced the vascular permeability and endothelial cell proliferation. In LPS-administered mice, the SFO vasculature showed a significant decrease in the immunoreactivity of plasmalemma vesicle associated protein-1, a marker of endothelial fenestral diaphragms. These data suggest that vasculature undergoes structural change to decrease vascular permeability in response to systemic LPS administration. © 2016 Published by Elsevier B.V.
1. Introduction The endotoxin lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria, acts as a potent inflammatory stimulus (West and Heagy, 2002). LPS stimulation results in an increase of proinflammatory cytokine release into the general circulation. Peripherally released proinflammatory cytokines act on the brain and this leads to the generation of fever (Kluger, 1991), the activation of the hypothalamic-pituitary-adrenal axis (Givalois et al., 1994), and many behavioral changes (Dantzer et al., 1998). However, direct access of LPS and cytokines to the brain is prevented by the blood–brain barrier (BBB) (Coceani et al., 1988). The BBB is stable in the adult mammalian brain except under pathological conditions such as injury or hypoxia (for a review, see Greenberg and Jin, 2005). The circumventricular organs (CVOs) are a group of small brain areas that lack a typical BBB. They contain a complex vascular plexus with fenestrated endothelial cells, enabling diffusion of circulating molecules into the parenchyma. The subfornical organ (SFO), one of the CVOs, contains neurons and is a key brain area for sensing circulating LPS and cytokines (Nadeau and Rivest, 2000; Quan et al., 1997). LPS
Abbreviations: BBB, blood–brain barrier; BrdU, 5-bromo-2′-deoxyuridine; CVO, circumventricular organ; Dex70k, Dextran 70,000; DSHB, Developmental Studies of Hybridoma Bank; FITC, fluorescein isothiocyanate; LPS, lipopolysaccharide; PB, phosphate buffer; PBS, phosphate-buffered saline; PFA, paraformaldehyde; PV-1, plasmalemma vesicle associated protein-1; SFO, subfornical organ. ⁎ Corresponding author. E-mail address: [email protected] (S. Morita-Takemura).
http://dx.doi.org/10.1016/j.jneuroim.2016.06.011 0165-5728/© 2016 Published by Elsevier B.V.
induces Fos expression in the rat SFO 2 to 3 h after intravenous injection (Sagar et al., 1995). Toll-like receptor-4 has been recognized as the main LPS-binding receptor (Hoshino et al., 1999; Poltorak et al., 1998), and we recently reported that Toll-like receptor-4 is expressed in glial fibrillary acidic protein-positive astrocytes and CD45-positive microglial cells/leukocytes in the SFO (Nakano et al., 2015). Another study demonstrated that electrolytic lesion of the SFO resulted in reduced LPS-induced fever in rats (Takahashi et al., 1997). We have also recently shown continuous angiogenesis in the CVOs of adult mice (Furube et al., 2014; Morita et al., 2015; Morita et al., 2013). Active proliferation of endothelial cells occurred in the CVOs, and the mitotic inhibitor cytosine arabinoside suppressed this proliferation of endothelial cells and reduced the vascular permeability of a peripherally administered tracer in the SFO (Morita et al., 2015). These data suggest that endothelial cell proliferation is closely correlated with vascular permeability in the SFO. Plasmalemma vesicle associated protein-1 (PV-1) encodes a transmembrane protein that is associated with the caveolae of fenestrated microvascular endothelial cells (Stan et al., 2004) and is therefore a useful marker of endothelial fenestral diaphragms (Ciofi et al., 2009; Ioannidou et al., 2006; Stan et al., 1999b). PV-1 is normally expressed in endothelial cells of the fetal brain (Engelhardt, 2003) and of the adult CVOs (Ciofi et al., 2009). PV-1 has been shown to be necessary for fenestral diaphragm formation in vitro (Stan et al., 2004). Moreover, the expression of PV-1 messenger RNA increased after fenestra formation in normally non-fenestrated human microvascular endothelial cells using tumor promoter, 4 beta-phorbol 12-myristate 13-acetate (Lombardi et al., 1986).
S. Morita-Takemura et al. / Journal of Neuroimmunology 298 (2016) 132–137
The aim of this study was to gain insight into the potential role of angiogenesis in the SFO during peripheral inflammation. To elucidate whether circulating LPS or cytokines change vascular permeability in the SFO, we performed a fluorescein isothiocyanate (FITC) and Dextran 70,000 (Dex70k) tracer assay after intraperitoneal LPS administration. In addition, we explored the effect of LPS administration on endothelial cell proliferation in the SFO using the mitotic marker 5-bromo-2′deoxyuridine (BrdU). Finally, we examined the expression level of PV1 using semi-quantitative morphometric analysis of PV-1 immunohistochemical data in the SFO of LPS-administered mice. 2. Materials and methods 2.1. Animals Adult male mice (C57BL/6J) of postnatal day 56–70 were used in the experiments. The animals were housed in a colony room with 12-h light/12-h dark cycle, and given access to commercial chow and tap water ad libitum. Animal care and experiments were conducted in accordance with the guidelines of the National Institutes of Health for the Care and Use of Laboratory Animals and the Guidelines for Proper Conduct of Animal Experiments of the Science Council of Japan. The Animal Care Committee of Nara Medical University approved the experimental protocol. 2.2. Administration of LPS and BrdU Mice were given a single intraperitoneal injection of LPS (1 mg/kg) from Escherichia coli (serotype 055:B5, Sigma-Aldrich Japan, Tokyo, Japan) in vehicle (pyrogen-free physiological saline (Ohtsuka Chemical, Tokushima, Japan)) according to a previous study (Monje et al., 2003). After LPS administration, the mice received an intraperitoneal injection of BrdU (Sigma-Aldrich Japan, 100 mg/kg/day) in phosphate-buffered saline (PBS; pH 7.2) twice daily for 4 days.
133
Thereafter, the sections were incubated with Alexa-488- or -594conjugated goat IgG against Armenian hamster, guinea pig, or rat IgG (Jackson ImmunoResearch, West Grove, PA; dilution 1:400) in PBST for 1 h. 2.4. Evaluation of vascular permeability using FITC Experimental administration and detection of FITC were performed according to our previous methods (Miyata and Morita, 2011; Morita et al., 2015). After anesthesia with pentobarbital, mice were perfused transcardially with oxygenated PBS (pH 7.0) containing 10 mM glucose for 1 min, and then with FITC (0.1 mg/ml; Dojindo, Tokyo, Japan) in oxygenated PBS (pH 7.0) containing 10 mM glucose for 3 min. Animals were then perfused transcardially with 4% PFA in 0.1 M PB (pH 8.0) for 2 min after a 1-min perfusion of oxygenated PBS (pH 7.0) containing 10 mM glucose. Dissected brains were postfixed in 4% PFA in 0.1 M PB (pH 8.0) for overnight at 4 °C and then kept in 30% sucrose in PBS (pH 8.0) for 24 h at 4 °C. Since FITC selectively binds primary amino groups and easily forms a stable covalent thiourea linkage (Mujumdar et al., 1989), the localization and intensity of FITC fluorescence is retained without dislocation or diffusion even after immunohistochemical procedures (Morita and Miyata, 2012). 2.5. Evaluation of vascular permeability using Dex70k Experimental administration and detection of Dex70k were performed according to our previous methods (Morita et al., 2016). Mice were subjected to intravenous administration (100 μl) of Texas-Redconjugated lysine-fixable Dex70k (MW = 70,000, Molecular Probes; 0.4 mg/ml) in PBS. Animals were sacrificed at 30 min after this administration for immunohistochemical analysis. They were then processed for immunohistochemistry. 2.6. Confocal observation
2.3. Light microscopic immunohistochemistry Mice were anesthetized by pentobarbital and perfused transcardially with PBS (pH 7.2), followed by 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB; pH 7.4). The dissected brains were postfixed with 4% PFA in 0.1 M PB (pH 7.4) overnight at 4 °C. They were then cryo-protected in buffered 30% sucrose for 24 h at 4 °C and frozen at − 80 °C in Tissue-Tec OCT compound (Sakura Finetechnical, Tokyo, Japan). Twenty-micrometer coronal sections were prepared using a cryostat microtome (CM1860, Leica, Wetzlar, Germany). For immunofluorescence detection, we processed free-floating sections as previously described (Morita et al., 2015). In brief, sections were washed with PBS, treated with 25 mM glycine in PBS for 30 min, incubated with 5% normal goat serum in PBS containing 0.3% Triton X100 (PBST) for 1 h, and then incubated with the following primary antibodies: Armenian hamster anti-CD31 (clone 2H8, DSHB; dilution 1:20) for 24 h at 4 °C, and guinea pig anti-laminin (YI-2008, Imamura et al. 2010; dilution 1:200) for 2 h at 37 °C. For PV-1 immunohistochemistry, mice were perfused with 1% PFA in 0.1 M PB (pH 7.4) for 2 min at 4 °C. Brains were dissected, postfixed in 1% PFA in 0.1 M PB (pH 7.4) at 4 °C overnight. The cryo-sections were mounted on slides coated with adhesive silane (Matsunami Glass, Osaka, Japan) and incubated with rat IgG anti-PV-1 (MECA-32, DSHB; dilution 1:100) for 48 h at 4 °C. For BrdU immunohistochemistry, sections were incubated in Na-citrate buffer (10 mM, pH 6.0) for 10 min at 95 °C, followed by 2 N HCl for 20 min at 37 °C. The sections were incubated with rat monoclonal IgG against BrdU (OBT0030, Serotec, Raleigh, NC; dilution 1:100) in PBST for 48 h at 4 °C.
Labeled sections were mounted on glass slides and coverslips were sealed with Vectashield (Vector Laboratories, Burlingame, CA). Stained sections were observed using laser-scanning confocal microscopes (LSM510, Carl Zeiss, Oberkochen, Germany or C2, Nikon Corporation, Tokyo, Japan). To minimize observation biases among different fields and sections, the parameters of the confocal microscope (such as pinhole size, brightness, and contrast setting) were maintained. We saved images (1024 × 1024 pixels) as TIFF files using Nikon Nis-Elements AR software and arranged them using Photoshop CS6. 2.7. Data analysis For semi-quantitative or quantitative analyses, the intensity of parenchymal FITC fluorescence and perivascular space Dex70k fluorescence, the area of PV-1 immunoreactivity, and the number of BrdUlabeled endothelial cells were measured in saved images using ImageJ software (National Institutes of Health). To measure the intensity of parenchymal FITC fluorescence and perivascular space Dex70k fluorescence, the laminin- or CD31-immunoreactive areas were pasted onto the tracer images to eliminate the fluorescence of the vascular area using Photoshop according to our previous study (Morita et al., 2014). Analysis of all images was performed such that the experimenter was blind to the treatment group. We used 3–5 animals from each group for quantification, and at least eight sections per animal were chosen from the SFO (between bregma − 0.46 and − 0.82 mm) and cerebral cortex (between bregma −0.46 and −0.82 mm) regions according to the mouse brain atlas (Paxinos and Franklin, 2001). Data are mean ±. Independent groups were compared with the Mann–Whitney U test. Difference was assessed at a significance level of P b 0.05.
134
S. Morita-Takemura et al. / Journal of Neuroimmunology 298 (2016) 132–137
3. Results 3.1. LPS decreases vascular permeability in the SFO To elucidate whether systemic administration of LPS changes the permeability of the vasculature in the SFO, we examined it using FITC after intraperitoneal LPS administration. We distinguished parenchymal FITC from circulating FITC using the vascular basement membrane marker laminin. The fluorescence intensity of parenchymal FITC in the SFO at 4 days was lower after intraperitoneal administration of LPS than after that of the vehicle control (Fig. 1). Semi-quantitative analysis revealed the decrease of the intensity of parenchymal FITC in the SFO after LPS administration (1 d, 52.8 ± 17.6%, p = 0.200, 4 d, 49.7 ± 4.99%, p = 0.029, Fig. 1D). In contrast, the fluorescence of parenchymal FITC remained unchanged after LPS administration in the cerebral cortex. The vascular permeability of higher molecular weight molecule was also examined with another fluorescent tracer Dex70k (MW: 70,000) (Fig. 2). Previously we demonstrated that blood-derived FITCbovine serum albumin (MW: 66,000–70,000) and Dextran 10,000 (MW: 10,000) remained in the perivascular space (Morita et al., 2016; Morita and Miyata, 2012). Computer-assisted images showed Dex70k
in the perivascular space of the SFO after vehicle administration (Fig. 2A1 and A3). In contrast, red fluorescence of perivascular space Dex70k decreased after LPS administration (Fig. 2B1 and B3). The fluorescence of Dex70k appeared closely associated with CD31-positive endothelial cells, often giving yellow colors (merged color of green and red; Fig. 2B2 and B4–B6). Semi-quantitative analyses revealed that the intensity of perivascular space Dex70k in the SFO after LPS administration was decreased as compared to the vehicle-administered animals (52.2 ± 6.52%, p = 0.029, Fig. 2C). These results indicate that vascular permeability decreases after LPS administration in the SFO of adult mice. 3.2. LPS attenuates endothelial cell proliferation in the SFO To determine whether proliferation of endothelial cells was affected by LPS administration, we performed BrdU immunohistochemistry in the SFO of adult mice. Double-labeling immunohistochemistry for BrdU and the endothelial cell marker CD31 revealed that BrdU-labeled nuclei were often detected in the vasculature of the SFO (Fig. 3A1 and A2), as previously reported (Morita et al., 2015). LPS administration apparently decreased the number of BrdU-labeled endothelial cells when
Fig. 1. Effect of LPS on the vascular permeability of circulating FITC in the adult SFO. Computer-assisted images of parenchymal FITC fluorescence (pFITC) and merged images of FITC and vascular basement membrane marker laminin immunoreactivity. A1–2 Typical confocal images showing FITC distribution in the SFO. B1–2, C1–2 Typical confocal images showing FITC distribution 1 d after (B1–2) or 4 d after (C1–2) intraperitoneal administration of LPS. D Semi-quantitative morphometric analysis revealing effects of LPS on the vascular permeability of circulating FITC in the SFO of adult mice. **P b 0.01, relative to vehicle, determined by the Mann–Whitney U test. Lam, laminin; 1 d, 1 day after LPS administration; 4 d, 4 days after LPS administration. Scale bar = 50 μm.
S. Morita-Takemura et al. / Journal of Neuroimmunology 298 (2016) 132–137
135
Fig. 2. Effect of LPS on the vascular permeability of circulating Dex70k in the adult SFO. A1–A6 Representative confocal images showing Dex70k distribution in the SFO of the vehicleadministered mice. B1–B6 Representative confocal images showing Dex70k distribution 4 d after intraperitoneal administration of LPS. Panel A1 and B1 shows computer-assisted perivascular red fluorescence of Dex70k (pDex70k) and A2 and B2 shows merged image of Dex70k and green fluorescence of CD-31 immunoreactivity. The panels in the right column (A3–A6 and B3–B6) represent the enlarged boxed areas of the left columns. Arrowheads indicate perivascular Dex70k and arrows indicate Dex70k closely associated with CD31. C Semi-quantitative morphometric analyses showing the effects of LPS on the vascular permeability of circulating Dex70k in the SFO of adult mice. P* b 0.05, relative to vehicle, determined by the Mann–Whitney U test. 4 d, 4 days after LPS administration. Scale bars = 50 (A1) and 10 (A3) μm.
compared with the vehicle control (Fig. 3B1 and B2), and semi-quantitative analysis revealed the significant decrease (62.4 ± 9.82, p = 0.029) (Fig. 3C). 3.3. LPS decreases the area of the PV-1-positive endothelial cells in the SFO Double-labeling immunohistochemistry showed that PV-1 immunoreactivity was present in CD31-positive endothelial cells (Fig. 4A1 and A2). LPS administration appeared to decrease the area of PV-1positive endothelial cells when compared with the vehicle control, especially in the outer shell of the SFO (Fig. 4B1 and B2). We confirmed by image analysis that the vasculature in LPS-administered mice displayed significant less PV-1 immunoreactivity (52.9 ± 4.03%, p = 0.032) than the vehicle control (76.1 ± 6.85%) at 4 d after LPS administration (Fig. 4C). 4. Discussion Dysfunction of the BBB can results in the accumulation of neurotoxic molecules within the brain parenchyma. The SFO, however, lacks the typical BBB. The SFO is a brain region well known for detecting
circulating LPS and cytokines and transducing information about them to other brain regions to control brain inflammatory responses. The main findings of the present study are as follows. First, single intraperitoneal administration of LPS significantly decreased FITC fluorescence in the parenchymal region and Dex70k fluorescence in the perivascular space of the SFO. Second, the number of BrdU-labeled CD31-positive endothelial cells decreased in LPS-administered mice. Finally, SFO capillaries showed a significant decrease in PV-1 immunoreactivity after LPS administration. These data suggest that the fenestrated vasculature of the SFO undergoes structural change to decrease vascular permeability after systemic LPS administration. Recently, it has been shown that fasting promotes endothelial cell fenestration in response to tanycytic vascular endothelial growth factor levels in one of the CVOs, the median eminence (Langlet et al., 2013). We have shown that chronic salt loading increased vascular permeability to FITC in some CVOs including the SFO of adult mice (Morita et al., 2014). In the present study, we found that LPS administration decreased the vascular permeability to FITC and Dex70k in the SFO. It is thus likely that vascular permeability not only increases but also decreases in response to peripheral signals in the CVOs.
136
S. Morita-Takemura et al. / Journal of Neuroimmunology 298 (2016) 132–137
Fig. 3. Effect of LPS administration on proliferation of endothelial cells in the adult SFO. BrdU-labeled endothelial cells (arrows in A1–2) were often seen in the SFO of vehicle-treated mice. LPS administration diminished BrdU labeling in endothelial cells (B1–2). Arrowheads indicate CD31-negative BrdU-labeled cells and arrows indicate CD31-positive BrdU-labeled cells. Scale bar = 50 μm. C Quantitative analysis revealing the effects of LPS on the percentage of BrdU-labeled endothelial cells in the SFO of adult mice. *P b 0.05 relative to vehicle, determined by the Mann–Whitney U test.
Quantitative analysis revealed that the administration of LPS markedly decreased the number of BrdU-labeled CD31-positive endothelial cells in the SFO. Vasculature is highly permeable and with continuous angiogenesis in the developing brain. Intraperitoneally injected rabbit IgG is found in the brain parenchyma of neonatal rats (Fabian and Hulsebosch, 1989) and proliferation of endothelial cells in the rat cerebral cortex is maximal at postnatal day 7 (Robertson et al., 1985). In our previous study, cytosine-b-D-arabinoside attenuated vascular permeability in the SFO (Morita et al., 2015). The present data suggest that physiological stimulation also changes the proliferation rate of endothelial cells and may result in the alteration of vascular permeability in the SFO. In contrast to proliferation of endothelial cells, many BrdU-labeled cells were present in the parenchyma of the SFO after LPS administration. Previous immunohistochemical analysis showed that BrdUlabeled nuclei were frequently observed in Iba1-positive myeloid lineage cells in the SFO of LPS-administered mice (Furube et al., 2015).
Fenestrae are specialized plasma membrane microdomains in endothelial cells that are involved in vascular permeability. PV-1 is a transmembrane protein associated with the caveolae of fenestrated microvascular endothelial cells (Stan et al., 1999a). Expression of PV-1 in endothelial cells gradually decreases as brain and vascular structures mature and is barely detectable by embryonic day 17 or at later stage of development in the brain except in the CVOs (Hallmann et al., 1995). Moreover, loss of integrity of the BBB results in the re-expression of PV-1 in the adult brain, as in the case of brain tumors and acute ischemia (Carson-Walter et al., 2005; Shue et al., 2008). These data suggest that PV-1 expression is highly correlated with vascular permeability in physiological and pathological conditions. We previously showed continuous angiogenesis occur in the adult SFO (Morita et al., 2015). We found high expression of vascular endothelial growth factor (VEGF) and its receptor VEGFR2 and proliferation of endothelial cells in the SFO. VEGF is involved in the induction or maintenance of endothelial
Fig. 4. Effect of LPS administration on PV-1 immunoreactivity in endothelial cells in the adult SFO. Representative confocal images show immunoreactivity for PV-1 and CD31 (A1–2). LPS administration decreased PV-1-positive endothelial cell area (B1–2). os outer shell of the SFO, vc ventromedial core of the SFO. Scale bar = 50 μm. C Semi-quantitative morphometric analysis revealing the effects of systemic LPS administration on PV-1 immunoreactivity in the SFO of adult mice. The ratio of PV-1/CD31 area decreased significantly after LPS administration relative to that of the vehicle. *P b 0.05 relative to vehicle, determined by the Mann–Whitney U test.
S. Morita-Takemura et al. / Journal of Neuroimmunology 298 (2016) 132–137
fenestrations. Topical administration of VEGF induced fenestrations in continuous microvascular endothelium of muscle and skin (Roberts and Palade, 1995). It has been shown that VEGF induces the expression of PV-1 and formation of caveolae and fenestrae in vitro (Strickland et al., 2005) (Esser et al., 1998). Moreover, recent study demonstrated that PV-1 inhibition attenuated VEGF-induced vascular permeability in blood–retinal barrier model (Wisniewska-Kruk et al., 2014; Wisniewska-Kruk et al., 2016). In the present study, expression of PV1 decreased in the SFO after LPS administration. PV-1 downregulation may thus underlie the mechanism of reduction of vascular permeability. However, we did not found significant expression changes of VEGF after LPS administration (data not shown). Further studies on the mechanisms by which LPS decreases the vascular permeability are warranted. Repeated exposure to LPS leads to attenuation of the fever response (Beeson, 1947). This phenomenon is known as endotoxin tolerance. In endotoxin tolerance, innate immune cells exposed to low concentrations of endotoxin enter a transient unresponsive state and are unable to respond to further challenges with endotoxin. Consequently, the production of circulating cytokines is reduced in endotoxin-tolerant animals (Erroi et al., 1993; Roth et al., 1994). In the brain, the CVOs are thought to regulate endotoxin tolerance. IL-1β production induced in the SFO by intravenous injection of LPS disappeared in LPS-tolerant rabbits (Nakamori et al., 1995). The decrease in vascular permeability in the SFO may have a role in endotoxin tolerance. Acknowledgements This work was supported in part by Scientific Research Grants from the Japan Society for the Promotion of Science (No. 26830029 to S. M. and Nos. 26293039 and 15K14354 to A. W.) and by a grant from the Takeda Science Foundation (JJ12010029 to A. W.). The hybridomas of anti-PV1 (MECA-32) IgG developed by Dr. Eugene C. Butcher and antiCD31 (2H8) antibody developed by Dr. Steven Bogen were obtained from the DSHB, developed under the auspices of the National Institute of Child Health and Human Development and maintained by the University of Iowa, Iowa City, IA 52242. References Beeson, P.B., 1947. Tolerance to bacterial pyrogens: I. Factors influencing its development. J. Exp. Med. 86, 29–38. Carson-Walter, E.B., Hampton, J., Shue, E., Geynisman, D.M., Pillai, P.K., Sathanoori, R., et al., 2005. Plasmalemmal vesicle associated protein-1 is a novel marker implicated in brain tumor angiogenesis. Clin. Cancer Res. 11, 7643–7650. Ciofi, P., Garret, M., Lapirot, O., Lafon, P., Loyens, A., Prevot, V., et al., 2009. Brain–endocrine interactions: a microvascular route in the mediobasal hypothalamus. Endocrinology 150, 5509–5519. Coceani, F., Lees, J., Dinarello, C.A., 1988. Occurrence of interleukin-1 in cerebrospinal fluid of the conscious cat. Brain Res. 446, 245–250. Dantzer, R., Bluthe, R.M., Gheusi, G., Cremona, S., Laye, S., Parnet, P., et al., 1998. Molecular basis of sickness behavior. Ann. N. Y. Acad. Sci. 856, 132–138. Engelhardt, B., 2003. Development of the blood–brain barrier. Cell Tissue Res. 314, 119–129. Erroi, A., Fantuzzi, G., Mengozzi, M., Sironi, M., Orencole, S.F., Clark, B.D., et al., 1993. Differential regulation of cytokine production in lipopolysaccharide tolerance in mice. Infect. Immun. 61, 4356–4359. Esser, S., Wolburg, K., Wolburg, H., Breier, G., Kurzchalia, T., Risau, W., 1998. Vascular endothelial growth factor induces endothelial fenestrations in vitro. J. Cell Biol. 140, 947–959. Fabian, R.H., Hulsebosch, C.E., 1989. Time course of penetration of xenogeneic IgG into the central nervous system of the neonatal rat: an immunohistochemical and radionuclide tracer study. J. Neuroimmunol. 24, 183–189. Furube, E., Mannari, T., Morita, S., Nishikawa, K., Yoshida, A., Itoh, M., et al., 2014. VEGFdependent and PDGF-dependent dynamic neurovascular reconstruction in the neurohypophysis of adult mice. J. Endocrinol. 222, 161–179. Furube, E., Morita, M., Miyata, S., 2015. Characterization of neural stem cells and their progeny in the sensory circumventricular organs of adult mouse. Cell Tissue Res. Givalois, L., Dornand, J., Mekaouche, M., Solier, M.D., Bristow, A.F., Ixart, G., et al., 1994. Temporal cascade of plasma level surges in acth, corticosterone, and cytokines in endotoxin-challenged rats. Am. J. Phys. 267, R164–R170. Greenberg, D.A., Jin, K., 2005. From angiogenesis to neuropathology. Nature 438, 954–959. Hallmann, R., Mayer, D.N., Berg, E.L., Broermann, R., Butcher, E.C., 1995. Novel mouse endothelial cell surface marker is suppressed during differentiation of the blood brain barrier. Dev. Dyn. 202, 325–332.
137
Hoshino, K., Takeuchi, O., Kawai, T., Sanjo, H., Ogawa, T., Takeda, Y., et al., 1999. Cutting edge: toll-like receptor 4 (tlr4)-deficient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4 as the LPS gene product. J. Immunol. 162, 3749–3752. Imamura, Y., Morita, S., Nakatani, Y., Okada, K., Ueshima, S., Matsuo, O., et al., 2010. Tissue plasminogen activator and plasminogen are critical for osmotic homeostasis by regulating vasopressin secretion. J. Neurosci. Res. 88, 1995–2006. Ioannidou, S., Deinhardt, K., Miotla, J., Bradley, J., Cheung, E., Samuelsson, S., et al., 2006. An in vitro assay reveals a role for the diaphragm protein PV-1 in endothelial fenestra morphogenesis. Proc. Natl. Acad. Sci. U. S. A. 103, 16770–16775. Kluger, M.J., 1991. Fever: role of pyrogens and cryogens. Physiol. Rev. 71, 93–127. Langlet, F., Levin, B.E., Luquet, S., Mazzone, M., Messina, A., Dunn-Meynell, A.A., et al., 2013. Tanycytic VEGF-a boosts blood–hypothalamus barrier plasticity and access of metabolic signals to the arcuate nucleus in response to fasting. Cell Metab. 17, 607–617. Lombardi, T., Montesano, R., Furie, M.B., Silverstein, S.C., Orci, L., 1986. Endothelial diaphragmed fenestrae: in vitro modulation by phorbol myristate acetate. J. Cell Biol. 102, 1965–1970. Miyata, S., Morita, S., 2011. A new method for visualization of endothelial cells and extravascular leakage in adult mouse brain using fluorescein isothiocyanate. J. Neurosci. Methods 202, 9–16. Monje, M.L., Toda, H., Palmer, T.D., 2003. Inflammatory blockade restores adult hippocampal neurogenesis. Science 302, 1760–1765. Morita, S., Miyata, S., 2012. Different vascular permeability between the sensory and secretory circumventricular organs of adult mouse brain. Cell Tissue Res. 349, 589–603. Morita, S., Ukai, S., Miyata, S., 2013. VEGF-dependent continuous angiogenesis in the median eminence of adult mice. Eur. J. Neurosci. 37, 508–518. Morita, S., Hourai, A., Miyata, S., 2014. Changes in pericytic expression of NG2 and PDGFRB and vascular permeability in the sensory circumventricular organs of adult mouse by osmotic stimulation. Cell Biochem. Funct. 32, 51–61. Morita, S., Furube, E., Mannari, T., Okuda, H., Tatsumi, K., Wanaka, A., et al., 2015. Vascular endothelial growth factor-dependent angiogenesis and dynamic vascular plasticity in the sensory circumventricular organs of adult mouse brain. Cell Tissue Res. 359, 865–884. Morita, S., Furube, E., Mannari, T., Okuda, H., Tatsumi, K., Wanaka, A., et al., 2016. Heterogeneous vascular permeability and alternative diffusion barrier in sensory circumventricular organs of adult mouse brain. Cell Tissue Res. 363, 497–511. Mujumdar, R.B., Ernst, L.A., Mujumdar, S.R., Waggoner, A.S., 1989. Cyanine dye labeling reagents containing isothiocyanate groups. Cytometry 10, 11–19. Nadeau, S., Rivest, S., 2000. Role of microglial-derived tumor necrosis factor in mediating CD14 transcription and nuclear factor kappa b activity in the brain during endotoxemia. J. Neurosci. 20, 3456–3468. Nakamori, T., Sakata, Y., Watanabe, T., Morimoto, A., Nakamura, S., Murakami, N., 1995. Suppression of interleukin-1 beta production in the circumventricular organs in endotoxin-tolerant rabbits. Brain Res. 675, 103–109. Nakano, Y., Furube, E., Morita, S., Wanaka, A., Nakashima, T., Miyata, S., 2015. Astrocytic TLR4 expression and LPS-induced nuclear translocation of STAT3 in the sensory circumventricular organs of adult mouse brain. J. Neuroimmunol. 278, 144–158. Paxinos, G., Franklin, K.B.J., 2001. The Mouse Brain in Stereotaxic Coordinates. second ed. Academic Press, San Diego. Poltorak, A., He, X., Smirnova, I., Liu, M.Y., Van Huffel, C., Du, X., et al., 1998. Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in TLR4 gene. Science 282, 2085–2088. Quan, N., Whiteside, M., Kim, L., Herkenham, M., 1997. Induction of inhibitory factor kappaBalpha mRNA in the central nervous system after peripheral lipopolysaccharide administration: an in situ hybridization histochemistry study in the rat. Proc. Natl. Acad. Sci. U. S. A. 94, 10985–10990. Roberts, W.G., Palade, G.E., 1995. Increased microvascular permeability and endothelial fenestration induced by vascular endothelial growth factor. J. Cell Sci. 108 (Pt 6), 2369–2379. Robertson, P.L., Du Bois, M., Bowman, P.D., Goldstein, G.W., 1985. Angiogenesis in developing rat brain: an in vivo and in vitro study. Brain Res. 355, 219–223. Roth, J., McClellan, J.L., Kluger, M.J., Zeisberger, E., 1994. Attenuation of fever and release of cytokines after repeated injections of lipopolysaccharide in guinea-pigs. J. Physiol. 477, 177–185. Sagar, S.M., Price, K.J., Kasting, N.W., Sharp, F.R., 1995. Anatomic patterns of fos immunostaining in rat brain following systemic endotoxin administration. Brain Res. Bull. 36, 381–392. Shue, E.H., Carson-Walter, E.B., Liu, Y., Winans, B.N., Ali, Z.S., Chen, J., et al., 2008. Plasmalemmal vesicle associated protein-1 (PV-1) is a marker of blood–brain barrier disruption in rodent models. BMC Neurosci. 9, 29. Stan, R.V., Ghitescu, L., Jacobson, B.S., Palade, G.E., 1999a. Isolation, cloning, and localization of rat PV-1, a novel endothelial caveolar protein. J. Cell Biol. 145, 1189–1198. Stan, R.V., Kubitza, M., Palade, G.E., 1999b. PV-1 is a component of the fenestral and stomatal diaphragms in fenestrated endothelia. Proc. Natl. Acad. Sci. U. S. A. 96, 13203–13207. Stan, R.V., Tkachenko, E., Niesman, I.R., 2004. PV1 is a key structural component for the formation of the stomatal and fenestral diaphragms. Mol. Biol. Cell 15, 3615–3630. Strickland, L.A., Jubb, A.M., Hongo, J.A., Zhong, F., Burwick, J., Fu, L., et al., 2005. Plasmalemmal vesicle-associated protein (PLVAP) is expressed by tumour endothelium and is upregulated by vascular endothelial growth factor-a (VEGF). J. Pathol. 206, 466–475. Takahashi, Y., Smith, P., Ferguson, A., Pittman, Q.J., 1997. Circumventricular organs and fever. Am. J. Phys. 273, R1690–R1695. West, M.A., Heagy, W., 2002. Endotoxin tolerance: a review. Crit. Care Med. 30, S64–S73. Wisniewska-Kruk, J., Klaassen, I., Vogels, I.M., Magno, A.L., Lai, C.M., Van Noorden, C.J., et al., 2014. Molecular analysis of blood–retinal barrier loss in the akimba mouse, a model of advanced diabetic retinopathy. Exp. Eye Res. 122, 123–131. Wisniewska-Kruk, J., van der Wijk, A.E., van Veen, H.A., Gorgels, T.G., Vogels, I.M., Versteeg, D., et al., 2016. Plasmalemma vesicle-associated protein has a key role in blood–retinal barrier loss. Am. J. Pathol. 186, 1044–1054.
Contents lists available at ScienceDirect
Journal of Neuroimmunology journal homepage: www.elsevier.com/locate/jneuroim
Changes in endothelial cell proliferation and vascular permeability after systemic lipopolysaccharide administration in the subfornical organ Shoko Morita-Takemura a,⁎, Kazuki Nakahara a, Kouko Tatsumi a, Hiroaki Okuda a,b, Tatsuhide Tanaka a, Ayami Isonishi a, Akio Wanaka a a b
Department of Anatomy & Neuroscience, Faculty of Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521, Japan Department of Functional Anatomy, Graduate School of Medical Science, Kanazawa University, Kanazawa, Japan
a r t i c l e
i n f o
Article history: Received 5 April 2016 Received in revised form 13 June 2016 Accepted 28 June 2016 Keywords: Angiogenesis Blood–brain barrier Fenestrated endothelial cells Inflammation
a b s t r a c t The subfornical organ (SFO) has highly permeable fenestrated vasculature and is a key site for immune-to-brain communications. Recently, we showed the occurrence of continuous angiogenesis in the SFO. In the present study, we found that systemic administration of bacterial lipopolysaccharide (LPS) reduced the vascular permeability and endothelial cell proliferation. In LPS-administered mice, the SFO vasculature showed a significant decrease in the immunoreactivity of plasmalemma vesicle associated protein-1, a marker of endothelial fenestral diaphragms. These data suggest that vasculature undergoes structural change to decrease vascular permeability in response to systemic LPS administration. © 2016 Published by Elsevier B.V.
1. Introduction The endotoxin lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria, acts as a potent inflammatory stimulus (West and Heagy, 2002). LPS stimulation results in an increase of proinflammatory cytokine release into the general circulation. Peripherally released proinflammatory cytokines act on the brain and this leads to the generation of fever (Kluger, 1991), the activation of the hypothalamic-pituitary-adrenal axis (Givalois et al., 1994), and many behavioral changes (Dantzer et al., 1998). However, direct access of LPS and cytokines to the brain is prevented by the blood–brain barrier (BBB) (Coceani et al., 1988). The BBB is stable in the adult mammalian brain except under pathological conditions such as injury or hypoxia (for a review, see Greenberg and Jin, 2005). The circumventricular organs (CVOs) are a group of small brain areas that lack a typical BBB. They contain a complex vascular plexus with fenestrated endothelial cells, enabling diffusion of circulating molecules into the parenchyma. The subfornical organ (SFO), one of the CVOs, contains neurons and is a key brain area for sensing circulating LPS and cytokines (Nadeau and Rivest, 2000; Quan et al., 1997). LPS
Abbreviations: BBB, blood–brain barrier; BrdU, 5-bromo-2′-deoxyuridine; CVO, circumventricular organ; Dex70k, Dextran 70,000; DSHB, Developmental Studies of Hybridoma Bank; FITC, fluorescein isothiocyanate; LPS, lipopolysaccharide; PB, phosphate buffer; PBS, phosphate-buffered saline; PFA, paraformaldehyde; PV-1, plasmalemma vesicle associated protein-1; SFO, subfornical organ. ⁎ Corresponding author. E-mail address: [email protected] (S. Morita-Takemura).
http://dx.doi.org/10.1016/j.jneuroim.2016.06.011 0165-5728/© 2016 Published by Elsevier B.V.
induces Fos expression in the rat SFO 2 to 3 h after intravenous injection (Sagar et al., 1995). Toll-like receptor-4 has been recognized as the main LPS-binding receptor (Hoshino et al., 1999; Poltorak et al., 1998), and we recently reported that Toll-like receptor-4 is expressed in glial fibrillary acidic protein-positive astrocytes and CD45-positive microglial cells/leukocytes in the SFO (Nakano et al., 2015). Another study demonstrated that electrolytic lesion of the SFO resulted in reduced LPS-induced fever in rats (Takahashi et al., 1997). We have also recently shown continuous angiogenesis in the CVOs of adult mice (Furube et al., 2014; Morita et al., 2015; Morita et al., 2013). Active proliferation of endothelial cells occurred in the CVOs, and the mitotic inhibitor cytosine arabinoside suppressed this proliferation of endothelial cells and reduced the vascular permeability of a peripherally administered tracer in the SFO (Morita et al., 2015). These data suggest that endothelial cell proliferation is closely correlated with vascular permeability in the SFO. Plasmalemma vesicle associated protein-1 (PV-1) encodes a transmembrane protein that is associated with the caveolae of fenestrated microvascular endothelial cells (Stan et al., 2004) and is therefore a useful marker of endothelial fenestral diaphragms (Ciofi et al., 2009; Ioannidou et al., 2006; Stan et al., 1999b). PV-1 is normally expressed in endothelial cells of the fetal brain (Engelhardt, 2003) and of the adult CVOs (Ciofi et al., 2009). PV-1 has been shown to be necessary for fenestral diaphragm formation in vitro (Stan et al., 2004). Moreover, the expression of PV-1 messenger RNA increased after fenestra formation in normally non-fenestrated human microvascular endothelial cells using tumor promoter, 4 beta-phorbol 12-myristate 13-acetate (Lombardi et al., 1986).
S. Morita-Takemura et al. / Journal of Neuroimmunology 298 (2016) 132–137
The aim of this study was to gain insight into the potential role of angiogenesis in the SFO during peripheral inflammation. To elucidate whether circulating LPS or cytokines change vascular permeability in the SFO, we performed a fluorescein isothiocyanate (FITC) and Dextran 70,000 (Dex70k) tracer assay after intraperitoneal LPS administration. In addition, we explored the effect of LPS administration on endothelial cell proliferation in the SFO using the mitotic marker 5-bromo-2′deoxyuridine (BrdU). Finally, we examined the expression level of PV1 using semi-quantitative morphometric analysis of PV-1 immunohistochemical data in the SFO of LPS-administered mice. 2. Materials and methods 2.1. Animals Adult male mice (C57BL/6J) of postnatal day 56–70 were used in the experiments. The animals were housed in a colony room with 12-h light/12-h dark cycle, and given access to commercial chow and tap water ad libitum. Animal care and experiments were conducted in accordance with the guidelines of the National Institutes of Health for the Care and Use of Laboratory Animals and the Guidelines for Proper Conduct of Animal Experiments of the Science Council of Japan. The Animal Care Committee of Nara Medical University approved the experimental protocol. 2.2. Administration of LPS and BrdU Mice were given a single intraperitoneal injection of LPS (1 mg/kg) from Escherichia coli (serotype 055:B5, Sigma-Aldrich Japan, Tokyo, Japan) in vehicle (pyrogen-free physiological saline (Ohtsuka Chemical, Tokushima, Japan)) according to a previous study (Monje et al., 2003). After LPS administration, the mice received an intraperitoneal injection of BrdU (Sigma-Aldrich Japan, 100 mg/kg/day) in phosphate-buffered saline (PBS; pH 7.2) twice daily for 4 days.
133
Thereafter, the sections were incubated with Alexa-488- or -594conjugated goat IgG against Armenian hamster, guinea pig, or rat IgG (Jackson ImmunoResearch, West Grove, PA; dilution 1:400) in PBST for 1 h. 2.4. Evaluation of vascular permeability using FITC Experimental administration and detection of FITC were performed according to our previous methods (Miyata and Morita, 2011; Morita et al., 2015). After anesthesia with pentobarbital, mice were perfused transcardially with oxygenated PBS (pH 7.0) containing 10 mM glucose for 1 min, and then with FITC (0.1 mg/ml; Dojindo, Tokyo, Japan) in oxygenated PBS (pH 7.0) containing 10 mM glucose for 3 min. Animals were then perfused transcardially with 4% PFA in 0.1 M PB (pH 8.0) for 2 min after a 1-min perfusion of oxygenated PBS (pH 7.0) containing 10 mM glucose. Dissected brains were postfixed in 4% PFA in 0.1 M PB (pH 8.0) for overnight at 4 °C and then kept in 30% sucrose in PBS (pH 8.0) for 24 h at 4 °C. Since FITC selectively binds primary amino groups and easily forms a stable covalent thiourea linkage (Mujumdar et al., 1989), the localization and intensity of FITC fluorescence is retained without dislocation or diffusion even after immunohistochemical procedures (Morita and Miyata, 2012). 2.5. Evaluation of vascular permeability using Dex70k Experimental administration and detection of Dex70k were performed according to our previous methods (Morita et al., 2016). Mice were subjected to intravenous administration (100 μl) of Texas-Redconjugated lysine-fixable Dex70k (MW = 70,000, Molecular Probes; 0.4 mg/ml) in PBS. Animals were sacrificed at 30 min after this administration for immunohistochemical analysis. They were then processed for immunohistochemistry. 2.6. Confocal observation
2.3. Light microscopic immunohistochemistry Mice were anesthetized by pentobarbital and perfused transcardially with PBS (pH 7.2), followed by 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB; pH 7.4). The dissected brains were postfixed with 4% PFA in 0.1 M PB (pH 7.4) overnight at 4 °C. They were then cryo-protected in buffered 30% sucrose for 24 h at 4 °C and frozen at − 80 °C in Tissue-Tec OCT compound (Sakura Finetechnical, Tokyo, Japan). Twenty-micrometer coronal sections were prepared using a cryostat microtome (CM1860, Leica, Wetzlar, Germany). For immunofluorescence detection, we processed free-floating sections as previously described (Morita et al., 2015). In brief, sections were washed with PBS, treated with 25 mM glycine in PBS for 30 min, incubated with 5% normal goat serum in PBS containing 0.3% Triton X100 (PBST) for 1 h, and then incubated with the following primary antibodies: Armenian hamster anti-CD31 (clone 2H8, DSHB; dilution 1:20) for 24 h at 4 °C, and guinea pig anti-laminin (YI-2008, Imamura et al. 2010; dilution 1:200) for 2 h at 37 °C. For PV-1 immunohistochemistry, mice were perfused with 1% PFA in 0.1 M PB (pH 7.4) for 2 min at 4 °C. Brains were dissected, postfixed in 1% PFA in 0.1 M PB (pH 7.4) at 4 °C overnight. The cryo-sections were mounted on slides coated with adhesive silane (Matsunami Glass, Osaka, Japan) and incubated with rat IgG anti-PV-1 (MECA-32, DSHB; dilution 1:100) for 48 h at 4 °C. For BrdU immunohistochemistry, sections were incubated in Na-citrate buffer (10 mM, pH 6.0) for 10 min at 95 °C, followed by 2 N HCl for 20 min at 37 °C. The sections were incubated with rat monoclonal IgG against BrdU (OBT0030, Serotec, Raleigh, NC; dilution 1:100) in PBST for 48 h at 4 °C.
Labeled sections were mounted on glass slides and coverslips were sealed with Vectashield (Vector Laboratories, Burlingame, CA). Stained sections were observed using laser-scanning confocal microscopes (LSM510, Carl Zeiss, Oberkochen, Germany or C2, Nikon Corporation, Tokyo, Japan). To minimize observation biases among different fields and sections, the parameters of the confocal microscope (such as pinhole size, brightness, and contrast setting) were maintained. We saved images (1024 × 1024 pixels) as TIFF files using Nikon Nis-Elements AR software and arranged them using Photoshop CS6. 2.7. Data analysis For semi-quantitative or quantitative analyses, the intensity of parenchymal FITC fluorescence and perivascular space Dex70k fluorescence, the area of PV-1 immunoreactivity, and the number of BrdUlabeled endothelial cells were measured in saved images using ImageJ software (National Institutes of Health). To measure the intensity of parenchymal FITC fluorescence and perivascular space Dex70k fluorescence, the laminin- or CD31-immunoreactive areas were pasted onto the tracer images to eliminate the fluorescence of the vascular area using Photoshop according to our previous study (Morita et al., 2014). Analysis of all images was performed such that the experimenter was blind to the treatment group. We used 3–5 animals from each group for quantification, and at least eight sections per animal were chosen from the SFO (between bregma − 0.46 and − 0.82 mm) and cerebral cortex (between bregma −0.46 and −0.82 mm) regions according to the mouse brain atlas (Paxinos and Franklin, 2001). Data are mean ±. Independent groups were compared with the Mann–Whitney U test. Difference was assessed at a significance level of P b 0.05.
134
S. Morita-Takemura et al. / Journal of Neuroimmunology 298 (2016) 132–137
3. Results 3.1. LPS decreases vascular permeability in the SFO To elucidate whether systemic administration of LPS changes the permeability of the vasculature in the SFO, we examined it using FITC after intraperitoneal LPS administration. We distinguished parenchymal FITC from circulating FITC using the vascular basement membrane marker laminin. The fluorescence intensity of parenchymal FITC in the SFO at 4 days was lower after intraperitoneal administration of LPS than after that of the vehicle control (Fig. 1). Semi-quantitative analysis revealed the decrease of the intensity of parenchymal FITC in the SFO after LPS administration (1 d, 52.8 ± 17.6%, p = 0.200, 4 d, 49.7 ± 4.99%, p = 0.029, Fig. 1D). In contrast, the fluorescence of parenchymal FITC remained unchanged after LPS administration in the cerebral cortex. The vascular permeability of higher molecular weight molecule was also examined with another fluorescent tracer Dex70k (MW: 70,000) (Fig. 2). Previously we demonstrated that blood-derived FITCbovine serum albumin (MW: 66,000–70,000) and Dextran 10,000 (MW: 10,000) remained in the perivascular space (Morita et al., 2016; Morita and Miyata, 2012). Computer-assisted images showed Dex70k
in the perivascular space of the SFO after vehicle administration (Fig. 2A1 and A3). In contrast, red fluorescence of perivascular space Dex70k decreased after LPS administration (Fig. 2B1 and B3). The fluorescence of Dex70k appeared closely associated with CD31-positive endothelial cells, often giving yellow colors (merged color of green and red; Fig. 2B2 and B4–B6). Semi-quantitative analyses revealed that the intensity of perivascular space Dex70k in the SFO after LPS administration was decreased as compared to the vehicle-administered animals (52.2 ± 6.52%, p = 0.029, Fig. 2C). These results indicate that vascular permeability decreases after LPS administration in the SFO of adult mice. 3.2. LPS attenuates endothelial cell proliferation in the SFO To determine whether proliferation of endothelial cells was affected by LPS administration, we performed BrdU immunohistochemistry in the SFO of adult mice. Double-labeling immunohistochemistry for BrdU and the endothelial cell marker CD31 revealed that BrdU-labeled nuclei were often detected in the vasculature of the SFO (Fig. 3A1 and A2), as previously reported (Morita et al., 2015). LPS administration apparently decreased the number of BrdU-labeled endothelial cells when
Fig. 1. Effect of LPS on the vascular permeability of circulating FITC in the adult SFO. Computer-assisted images of parenchymal FITC fluorescence (pFITC) and merged images of FITC and vascular basement membrane marker laminin immunoreactivity. A1–2 Typical confocal images showing FITC distribution in the SFO. B1–2, C1–2 Typical confocal images showing FITC distribution 1 d after (B1–2) or 4 d after (C1–2) intraperitoneal administration of LPS. D Semi-quantitative morphometric analysis revealing effects of LPS on the vascular permeability of circulating FITC in the SFO of adult mice. **P b 0.01, relative to vehicle, determined by the Mann–Whitney U test. Lam, laminin; 1 d, 1 day after LPS administration; 4 d, 4 days after LPS administration. Scale bar = 50 μm.
S. Morita-Takemura et al. / Journal of Neuroimmunology 298 (2016) 132–137
135
Fig. 2. Effect of LPS on the vascular permeability of circulating Dex70k in the adult SFO. A1–A6 Representative confocal images showing Dex70k distribution in the SFO of the vehicleadministered mice. B1–B6 Representative confocal images showing Dex70k distribution 4 d after intraperitoneal administration of LPS. Panel A1 and B1 shows computer-assisted perivascular red fluorescence of Dex70k (pDex70k) and A2 and B2 shows merged image of Dex70k and green fluorescence of CD-31 immunoreactivity. The panels in the right column (A3–A6 and B3–B6) represent the enlarged boxed areas of the left columns. Arrowheads indicate perivascular Dex70k and arrows indicate Dex70k closely associated with CD31. C Semi-quantitative morphometric analyses showing the effects of LPS on the vascular permeability of circulating Dex70k in the SFO of adult mice. P* b 0.05, relative to vehicle, determined by the Mann–Whitney U test. 4 d, 4 days after LPS administration. Scale bars = 50 (A1) and 10 (A3) μm.
compared with the vehicle control (Fig. 3B1 and B2), and semi-quantitative analysis revealed the significant decrease (62.4 ± 9.82, p = 0.029) (Fig. 3C). 3.3. LPS decreases the area of the PV-1-positive endothelial cells in the SFO Double-labeling immunohistochemistry showed that PV-1 immunoreactivity was present in CD31-positive endothelial cells (Fig. 4A1 and A2). LPS administration appeared to decrease the area of PV-1positive endothelial cells when compared with the vehicle control, especially in the outer shell of the SFO (Fig. 4B1 and B2). We confirmed by image analysis that the vasculature in LPS-administered mice displayed significant less PV-1 immunoreactivity (52.9 ± 4.03%, p = 0.032) than the vehicle control (76.1 ± 6.85%) at 4 d after LPS administration (Fig. 4C). 4. Discussion Dysfunction of the BBB can results in the accumulation of neurotoxic molecules within the brain parenchyma. The SFO, however, lacks the typical BBB. The SFO is a brain region well known for detecting
circulating LPS and cytokines and transducing information about them to other brain regions to control brain inflammatory responses. The main findings of the present study are as follows. First, single intraperitoneal administration of LPS significantly decreased FITC fluorescence in the parenchymal region and Dex70k fluorescence in the perivascular space of the SFO. Second, the number of BrdU-labeled CD31-positive endothelial cells decreased in LPS-administered mice. Finally, SFO capillaries showed a significant decrease in PV-1 immunoreactivity after LPS administration. These data suggest that the fenestrated vasculature of the SFO undergoes structural change to decrease vascular permeability after systemic LPS administration. Recently, it has been shown that fasting promotes endothelial cell fenestration in response to tanycytic vascular endothelial growth factor levels in one of the CVOs, the median eminence (Langlet et al., 2013). We have shown that chronic salt loading increased vascular permeability to FITC in some CVOs including the SFO of adult mice (Morita et al., 2014). In the present study, we found that LPS administration decreased the vascular permeability to FITC and Dex70k in the SFO. It is thus likely that vascular permeability not only increases but also decreases in response to peripheral signals in the CVOs.
136
S. Morita-Takemura et al. / Journal of Neuroimmunology 298 (2016) 132–137
Fig. 3. Effect of LPS administration on proliferation of endothelial cells in the adult SFO. BrdU-labeled endothelial cells (arrows in A1–2) were often seen in the SFO of vehicle-treated mice. LPS administration diminished BrdU labeling in endothelial cells (B1–2). Arrowheads indicate CD31-negative BrdU-labeled cells and arrows indicate CD31-positive BrdU-labeled cells. Scale bar = 50 μm. C Quantitative analysis revealing the effects of LPS on the percentage of BrdU-labeled endothelial cells in the SFO of adult mice. *P b 0.05 relative to vehicle, determined by the Mann–Whitney U test.
Quantitative analysis revealed that the administration of LPS markedly decreased the number of BrdU-labeled CD31-positive endothelial cells in the SFO. Vasculature is highly permeable and with continuous angiogenesis in the developing brain. Intraperitoneally injected rabbit IgG is found in the brain parenchyma of neonatal rats (Fabian and Hulsebosch, 1989) and proliferation of endothelial cells in the rat cerebral cortex is maximal at postnatal day 7 (Robertson et al., 1985). In our previous study, cytosine-b-D-arabinoside attenuated vascular permeability in the SFO (Morita et al., 2015). The present data suggest that physiological stimulation also changes the proliferation rate of endothelial cells and may result in the alteration of vascular permeability in the SFO. In contrast to proliferation of endothelial cells, many BrdU-labeled cells were present in the parenchyma of the SFO after LPS administration. Previous immunohistochemical analysis showed that BrdUlabeled nuclei were frequently observed in Iba1-positive myeloid lineage cells in the SFO of LPS-administered mice (Furube et al., 2015).
Fenestrae are specialized plasma membrane microdomains in endothelial cells that are involved in vascular permeability. PV-1 is a transmembrane protein associated with the caveolae of fenestrated microvascular endothelial cells (Stan et al., 1999a). Expression of PV-1 in endothelial cells gradually decreases as brain and vascular structures mature and is barely detectable by embryonic day 17 or at later stage of development in the brain except in the CVOs (Hallmann et al., 1995). Moreover, loss of integrity of the BBB results in the re-expression of PV-1 in the adult brain, as in the case of brain tumors and acute ischemia (Carson-Walter et al., 2005; Shue et al., 2008). These data suggest that PV-1 expression is highly correlated with vascular permeability in physiological and pathological conditions. We previously showed continuous angiogenesis occur in the adult SFO (Morita et al., 2015). We found high expression of vascular endothelial growth factor (VEGF) and its receptor VEGFR2 and proliferation of endothelial cells in the SFO. VEGF is involved in the induction or maintenance of endothelial
Fig. 4. Effect of LPS administration on PV-1 immunoreactivity in endothelial cells in the adult SFO. Representative confocal images show immunoreactivity for PV-1 and CD31 (A1–2). LPS administration decreased PV-1-positive endothelial cell area (B1–2). os outer shell of the SFO, vc ventromedial core of the SFO. Scale bar = 50 μm. C Semi-quantitative morphometric analysis revealing the effects of systemic LPS administration on PV-1 immunoreactivity in the SFO of adult mice. The ratio of PV-1/CD31 area decreased significantly after LPS administration relative to that of the vehicle. *P b 0.05 relative to vehicle, determined by the Mann–Whitney U test.
S. Morita-Takemura et al. / Journal of Neuroimmunology 298 (2016) 132–137
fenestrations. Topical administration of VEGF induced fenestrations in continuous microvascular endothelium of muscle and skin (Roberts and Palade, 1995). It has been shown that VEGF induces the expression of PV-1 and formation of caveolae and fenestrae in vitro (Strickland et al., 2005) (Esser et al., 1998). Moreover, recent study demonstrated that PV-1 inhibition attenuated VEGF-induced vascular permeability in blood–retinal barrier model (Wisniewska-Kruk et al., 2014; Wisniewska-Kruk et al., 2016). In the present study, expression of PV1 decreased in the SFO after LPS administration. PV-1 downregulation may thus underlie the mechanism of reduction of vascular permeability. However, we did not found significant expression changes of VEGF after LPS administration (data not shown). Further studies on the mechanisms by which LPS decreases the vascular permeability are warranted. Repeated exposure to LPS leads to attenuation of the fever response (Beeson, 1947). This phenomenon is known as endotoxin tolerance. In endotoxin tolerance, innate immune cells exposed to low concentrations of endotoxin enter a transient unresponsive state and are unable to respond to further challenges with endotoxin. Consequently, the production of circulating cytokines is reduced in endotoxin-tolerant animals (Erroi et al., 1993; Roth et al., 1994). In the brain, the CVOs are thought to regulate endotoxin tolerance. IL-1β production induced in the SFO by intravenous injection of LPS disappeared in LPS-tolerant rabbits (Nakamori et al., 1995). The decrease in vascular permeability in the SFO may have a role in endotoxin tolerance. Acknowledgements This work was supported in part by Scientific Research Grants from the Japan Society for the Promotion of Science (No. 26830029 to S. M. and Nos. 26293039 and 15K14354 to A. W.) and by a grant from the Takeda Science Foundation (JJ12010029 to A. W.). The hybridomas of anti-PV1 (MECA-32) IgG developed by Dr. Eugene C. Butcher and antiCD31 (2H8) antibody developed by Dr. Steven Bogen were obtained from the DSHB, developed under the auspices of the National Institute of Child Health and Human Development and maintained by the University of Iowa, Iowa City, IA 52242. References Beeson, P.B., 1947. Tolerance to bacterial pyrogens: I. Factors influencing its development. J. Exp. Med. 86, 29–38. Carson-Walter, E.B., Hampton, J., Shue, E., Geynisman, D.M., Pillai, P.K., Sathanoori, R., et al., 2005. Plasmalemmal vesicle associated protein-1 is a novel marker implicated in brain tumor angiogenesis. Clin. Cancer Res. 11, 7643–7650. Ciofi, P., Garret, M., Lapirot, O., Lafon, P., Loyens, A., Prevot, V., et al., 2009. Brain–endocrine interactions: a microvascular route in the mediobasal hypothalamus. Endocrinology 150, 5509–5519. Coceani, F., Lees, J., Dinarello, C.A., 1988. Occurrence of interleukin-1 in cerebrospinal fluid of the conscious cat. Brain Res. 446, 245–250. Dantzer, R., Bluthe, R.M., Gheusi, G., Cremona, S., Laye, S., Parnet, P., et al., 1998. Molecular basis of sickness behavior. Ann. N. Y. Acad. Sci. 856, 132–138. Engelhardt, B., 2003. Development of the blood–brain barrier. Cell Tissue Res. 314, 119–129. Erroi, A., Fantuzzi, G., Mengozzi, M., Sironi, M., Orencole, S.F., Clark, B.D., et al., 1993. Differential regulation of cytokine production in lipopolysaccharide tolerance in mice. Infect. Immun. 61, 4356–4359. Esser, S., Wolburg, K., Wolburg, H., Breier, G., Kurzchalia, T., Risau, W., 1998. Vascular endothelial growth factor induces endothelial fenestrations in vitro. J. Cell Biol. 140, 947–959. Fabian, R.H., Hulsebosch, C.E., 1989. Time course of penetration of xenogeneic IgG into the central nervous system of the neonatal rat: an immunohistochemical and radionuclide tracer study. J. Neuroimmunol. 24, 183–189. Furube, E., Mannari, T., Morita, S., Nishikawa, K., Yoshida, A., Itoh, M., et al., 2014. VEGFdependent and PDGF-dependent dynamic neurovascular reconstruction in the neurohypophysis of adult mice. J. Endocrinol. 222, 161–179. Furube, E., Morita, M., Miyata, S., 2015. Characterization of neural stem cells and their progeny in the sensory circumventricular organs of adult mouse. Cell Tissue Res. Givalois, L., Dornand, J., Mekaouche, M., Solier, M.D., Bristow, A.F., Ixart, G., et al., 1994. Temporal cascade of plasma level surges in acth, corticosterone, and cytokines in endotoxin-challenged rats. Am. J. Phys. 267, R164–R170. Greenberg, D.A., Jin, K., 2005. From angiogenesis to neuropathology. Nature 438, 954–959. Hallmann, R., Mayer, D.N., Berg, E.L., Broermann, R., Butcher, E.C., 1995. Novel mouse endothelial cell surface marker is suppressed during differentiation of the blood brain barrier. Dev. Dyn. 202, 325–332.
137
Hoshino, K., Takeuchi, O., Kawai, T., Sanjo, H., Ogawa, T., Takeda, Y., et al., 1999. Cutting edge: toll-like receptor 4 (tlr4)-deficient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4 as the LPS gene product. J. Immunol. 162, 3749–3752. Imamura, Y., Morita, S., Nakatani, Y., Okada, K., Ueshima, S., Matsuo, O., et al., 2010. Tissue plasminogen activator and plasminogen are critical for osmotic homeostasis by regulating vasopressin secretion. J. Neurosci. Res. 88, 1995–2006. Ioannidou, S., Deinhardt, K., Miotla, J., Bradley, J., Cheung, E., Samuelsson, S., et al., 2006. An in vitro assay reveals a role for the diaphragm protein PV-1 in endothelial fenestra morphogenesis. Proc. Natl. Acad. Sci. U. S. A. 103, 16770–16775. Kluger, M.J., 1991. Fever: role of pyrogens and cryogens. Physiol. Rev. 71, 93–127. Langlet, F., Levin, B.E., Luquet, S., Mazzone, M., Messina, A., Dunn-Meynell, A.A., et al., 2013. Tanycytic VEGF-a boosts blood–hypothalamus barrier plasticity and access of metabolic signals to the arcuate nucleus in response to fasting. Cell Metab. 17, 607–617. Lombardi, T., Montesano, R., Furie, M.B., Silverstein, S.C., Orci, L., 1986. Endothelial diaphragmed fenestrae: in vitro modulation by phorbol myristate acetate. J. Cell Biol. 102, 1965–1970. Miyata, S., Morita, S., 2011. A new method for visualization of endothelial cells and extravascular leakage in adult mouse brain using fluorescein isothiocyanate. J. Neurosci. Methods 202, 9–16. Monje, M.L., Toda, H., Palmer, T.D., 2003. Inflammatory blockade restores adult hippocampal neurogenesis. Science 302, 1760–1765. Morita, S., Miyata, S., 2012. Different vascular permeability between the sensory and secretory circumventricular organs of adult mouse brain. Cell Tissue Res. 349, 589–603. Morita, S., Ukai, S., Miyata, S., 2013. VEGF-dependent continuous angiogenesis in the median eminence of adult mice. Eur. J. Neurosci. 37, 508–518. Morita, S., Hourai, A., Miyata, S., 2014. Changes in pericytic expression of NG2 and PDGFRB and vascular permeability in the sensory circumventricular organs of adult mouse by osmotic stimulation. Cell Biochem. Funct. 32, 51–61. Morita, S., Furube, E., Mannari, T., Okuda, H., Tatsumi, K., Wanaka, A., et al., 2015. Vascular endothelial growth factor-dependent angiogenesis and dynamic vascular plasticity in the sensory circumventricular organs of adult mouse brain. Cell Tissue Res. 359, 865–884. Morita, S., Furube, E., Mannari, T., Okuda, H., Tatsumi, K., Wanaka, A., et al., 2016. Heterogeneous vascular permeability and alternative diffusion barrier in sensory circumventricular organs of adult mouse brain. Cell Tissue Res. 363, 497–511. Mujumdar, R.B., Ernst, L.A., Mujumdar, S.R., Waggoner, A.S., 1989. Cyanine dye labeling reagents containing isothiocyanate groups. Cytometry 10, 11–19. Nadeau, S., Rivest, S., 2000. Role of microglial-derived tumor necrosis factor in mediating CD14 transcription and nuclear factor kappa b activity in the brain during endotoxemia. J. Neurosci. 20, 3456–3468. Nakamori, T., Sakata, Y., Watanabe, T., Morimoto, A., Nakamura, S., Murakami, N., 1995. Suppression of interleukin-1 beta production in the circumventricular organs in endotoxin-tolerant rabbits. Brain Res. 675, 103–109. Nakano, Y., Furube, E., Morita, S., Wanaka, A., Nakashima, T., Miyata, S., 2015. Astrocytic TLR4 expression and LPS-induced nuclear translocation of STAT3 in the sensory circumventricular organs of adult mouse brain. J. Neuroimmunol. 278, 144–158. Paxinos, G., Franklin, K.B.J., 2001. The Mouse Brain in Stereotaxic Coordinates. second ed. Academic Press, San Diego. Poltorak, A., He, X., Smirnova, I., Liu, M.Y., Van Huffel, C., Du, X., et al., 1998. Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in TLR4 gene. Science 282, 2085–2088. Quan, N., Whiteside, M., Kim, L., Herkenham, M., 1997. Induction of inhibitory factor kappaBalpha mRNA in the central nervous system after peripheral lipopolysaccharide administration: an in situ hybridization histochemistry study in the rat. Proc. Natl. Acad. Sci. U. S. A. 94, 10985–10990. Roberts, W.G., Palade, G.E., 1995. Increased microvascular permeability and endothelial fenestration induced by vascular endothelial growth factor. J. Cell Sci. 108 (Pt 6), 2369–2379. Robertson, P.L., Du Bois, M., Bowman, P.D., Goldstein, G.W., 1985. Angiogenesis in developing rat brain: an in vivo and in vitro study. Brain Res. 355, 219–223. Roth, J., McClellan, J.L., Kluger, M.J., Zeisberger, E., 1994. Attenuation of fever and release of cytokines after repeated injections of lipopolysaccharide in guinea-pigs. J. Physiol. 477, 177–185. Sagar, S.M., Price, K.J., Kasting, N.W., Sharp, F.R., 1995. Anatomic patterns of fos immunostaining in rat brain following systemic endotoxin administration. Brain Res. Bull. 36, 381–392. Shue, E.H., Carson-Walter, E.B., Liu, Y., Winans, B.N., Ali, Z.S., Chen, J., et al., 2008. Plasmalemmal vesicle associated protein-1 (PV-1) is a marker of blood–brain barrier disruption in rodent models. BMC Neurosci. 9, 29. Stan, R.V., Ghitescu, L., Jacobson, B.S., Palade, G.E., 1999a. Isolation, cloning, and localization of rat PV-1, a novel endothelial caveolar protein. J. Cell Biol. 145, 1189–1198. Stan, R.V., Kubitza, M., Palade, G.E., 1999b. PV-1 is a component of the fenestral and stomatal diaphragms in fenestrated endothelia. Proc. Natl. Acad. Sci. U. S. A. 96, 13203–13207. Stan, R.V., Tkachenko, E., Niesman, I.R., 2004. PV1 is a key structural component for the formation of the stomatal and fenestral diaphragms. Mol. Biol. Cell 15, 3615–3630. Strickland, L.A., Jubb, A.M., Hongo, J.A., Zhong, F., Burwick, J., Fu, L., et al., 2005. Plasmalemmal vesicle-associated protein (PLVAP) is expressed by tumour endothelium and is upregulated by vascular endothelial growth factor-a (VEGF). J. Pathol. 206, 466–475. Takahashi, Y., Smith, P., Ferguson, A., Pittman, Q.J., 1997. Circumventricular organs and fever. Am. J. Phys. 273, R1690–R1695. West, M.A., Heagy, W., 2002. Endotoxin tolerance: a review. Crit. Care Med. 30, S64–S73. Wisniewska-Kruk, J., Klaassen, I., Vogels, I.M., Magno, A.L., Lai, C.M., Van Noorden, C.J., et al., 2014. Molecular analysis of blood–retinal barrier loss in the akimba mouse, a model of advanced diabetic retinopathy. Exp. Eye Res. 122, 123–131. Wisniewska-Kruk, J., van der Wijk, A.E., van Veen, H.A., Gorgels, T.G., Vogels, I.M., Versteeg, D., et al., 2016. Plasmalemma vesicle-associated protein has a key role in blood–retinal barrier loss. Am. J. Pathol. 186, 1044–1054.