Vascular insufficiency,not inflammation,contributes to chronic gliosis in a rat CNS transplantation model

Abstract

Purpose: There is considerable variability in the extent and nature of the glial response to injury and neurodegeneration. Transplantation of fetal cortical tissue onto the brain of neonatal host rats or mice results in region-specific changes dependent on where the fetal tissue is placed. These changes include chronic astrocytic and microglial gliosis, oxidative stress, and altered metabolism of a number of proteins associated with the pathogenesis of Alzheimer’s disease. Such changes are only observed in heterotopic (cortex-to-midbrain) grafts and are not observed in homotopic cortex-to-cortex grafts. We investigated two possible triggers for the region-specific gliosis observed in our transplant model hypothesizing that either i) poor vascularization and lack of blood brain barrier integrity or ii) an inflammatory response initiated by the transplantation process, contributed to establishing chronic pathological changes.

Methods: We analyzed the time course of neovascularization, blood brain barrier permeability and inflammation using a combination of immunohistochemistry, enzyme-linked immunosorbant assay and Evan’s blue dye extravasation techniques.

Results: Blood brain barrier permeability and altered neovascularization occurred prior to the onset of gliosis in heterotopic grafts.

Conclusion: These data suggest that ischemic conditions and blood brain barrier damage can be a primary mechanism that initiates chronic gliosis and associated inflammatory changes in central nervous system tissue.

Keywords

Astrocytes transplantation inflammation blood brain barrier gliosis neovascularization

1 Introduction

Astrocytes become activated in response to many forms of brain injury and disease, in a process generally known as reactive astrogliosis. After injury to the central nervous system (CNS), the progression of initial astrocyte activation and proliferation through to the physical formation of a glial scar and the deposition of growth-inhibitory extracellular matrix molecules such as chondroitin sulfate proteoglycans (CSPG) is a major impediment to axon regeneration (Fitch et al., 2008; Rolls et al., 2009). However the modern definition of reactive astrogliosis also recognizes that this phenomenon can have beneficial as well as detrimental effects on surrounding tissue (Bush et al., 1999; Sofroniew, 2005, 2014; Li et al., 2008; Burda et al., 2014, 2016).

Several studies have demonstrated that reactive astrogliosis is modifiable (Gadea et al., 2008; Goldshmit et al., 2010; Sahni et al., 2010) and that activated astrocytes secrete a range of factors capable of promoting and sustaining neuronal repair, thus highlighting the exciting and realistic potential of glial-modulation as a neurotherapeutic strategy (Sofroniew, 2009). Such strategies may need to be tailored to particular circumstances because astrocytes display considerable heterogeneity in terms of their gene expression profile, morphology, function and reactivity (Rusnakova et al., 2013). Thus the timing and context of any intervention to alter glial reactivity after neurotrauma may be a critically important consideration when designing therapeutic regimes (Colangelo et al., 2014; Pekny et al., 2014). The response of astrocytes to injury differs according to the type of insult (e.g. stroke versus inflammation) (Zamanian et al., 2012), although the JAK2-STAT3 signaling cascade does appear to be consistently associated with glial fibrillary acid protein (GFAP) expression, astrocyte activation and glial scar formation (Herrmann et al., 2008; O’Callaghan et al., 2014). Cytokines and growth factors, including transforming growth factor-β1 (TGF-β1) and interleukin-6 (IL-6) are also important in the initiation and propagation of chronic astrogliosis through their respective signaling pathways (SMAD3 and STAT3) (Herrmann et al., 2008; Hamby et al., 2010; Su et al., 2010).

Given the complex and context-dependent response of astrocytes to injury, it is important to develop a variety of models of reactive gliosis to better understand the role of this process in the context of neuroprotection and repair (Burda et al., 2014). We have developed a unique fetal transplantation model of chronic cortical gliosis. Transplantation of fetal cortical tissue onto the midbrain region of neonatal hosts (heterotopic transplants) results in chronic activation of astrocytes and microglia with seemingly no effect on oligodendroglia (Majda et al., 1989; Harvey et al., 1997). Chronic activation of astrocytes and microglia in this model is associated with oxidative stress (Bates et al., 2007), deposition of CSPG and altered extracellular diffusion properties (Sykova et al., 1999), and increased expression of a number of proteins associated with Alzheimer’s disease (AD) (Martins et al., 2001; Bates et al., 2002).

This gliotic reaction is sustained for many months, and is seen in various rat strains (Harvey et al., 1997; Sykova et al., 1999) and in mice (Bates et al., 2002), but does not occur when similar fetal cortical tissue is transplanted onto the cerebral cortex of neonatal animals (homotopic transplants). There is also no significant gliosis in underlying host tissue. This model is therefore unique in that we observe reactive changes that are maintained for many months, potentially allowing us to investigate the sequence of neuropathological events that cause, and are then triggered by, reactive gliosis.

Because astrocytes are known to play an important role in the development and maintenance of the blood brain barrier (BBB) and are responsive to ischemic conditions (Petito et al., 1992; Li et al., 2008; Chisholm et al., 2016), we hypothesized that poor vascularization and lack of BBB integrity may underlie the region-specific effects seen after heterotopic transplantation. We thus investigated the permeability of transplanted tissue to Evan’s Blue Dye (EBD) at various time-points post-transplantation and determined the vascularity of grafted tissue using immunohistochemical techniques. Because pro-inflammatory cytokines are known to initiate reactive gliosis and modulate astrocyte signaling (Sofroniew, 2014), we also compared the time-course and extent of inflammatory cytokine expression in heterotopic and homotopic cortical grafts using enzyme-linked immunosorbant assay (ELISA) methods.

2 Materials and methods

Animal experimentation was performed with the approval of the Animal Ethics Committee of The University of Western Australia (approval number 01/100/194) according to National Health and Medical Research (NHMRC) Australian guidelines. Rats were provided by the Animal Resources Centre (WA, Australia). The transplantation procedure in neonatal rats has been published previously (Harvey et al., 1997; Sykova et al., 1999; Martins et al., 2001; Bates et al., 2007).

To obtain donor tissue and neonatal hosts of the appropriate age, mothers were time-mated so that host litters were born (P0) the same day that donor litters were embryonic (E) age 15-16 (day of fertilization = E0). Pregnant mothers were anaesthetized with 5% halothane, all embryos removed and quickly placed in ice-cold Hams F-10 media. The donor dams were then euthanized with an overdose of sodium pentobarbitone (Lethabarb, Virbac, NSW Australia, 150 mg/kg). Cortical tissue (specifically neocortical neuroepithelium approximately 150–200μm thick) was dissected from the cerebral hemispheres of donor embryos, overlying meningeal membranes removed and the tissue dissected into pieces approximately 2 mm². The grafts consisted of cortical tissue from the ventricular through to the pial surfaces and contained presumptive grey and white matter.

Neonatal pups were anaesthetized with ether and a small incision made through the skin and skull. A piece of cortical material was slowly injected under the cranium onto the dorsal surface of the left midbrain or onto the medial part of the right cortex (1-2 mm anterior to lambda) via a glass micropipette attached to a Hamilton syringe (Sykova et al., 1999; Martins et al., 2001). This approach leaves the cranium intact and substantially reduces the loss of tissue after the glass pipette is removed (Lund et al., 1981). Pups were then warmed before being returned to their mothers. The total number of rats used for each experimental method is outlined in Table 1 and the mortality rate for this method of transplantation was zero.

Long-term astrogliosis in fetal cortex transplanted heterotopically onto neonatal host midbrain is consistently seen irrespective of the species (rat e.g. (Bates et al., 2007), mouse e.g. (Bates et al., 2002)) or strain of rat (e.g. PVG/c (Harvey et al., 1997; Sykova et al., 1999), Wistar (Krum et al., 1989)) that is used. Here we used two inbred rat strains because of limited availability of animals at the time: F344 rats were used for studies related to inflammation and PVG/c rats for the vascularization studies. Animals were euthanized with an i.p. injection of sodium pentobarbitone (Lethabarb, Virbac, NSW Australia, 150 mg/kg) prior to tissue removal. We chose to focus on three time points; 2 weeks, 4 weeks and 3 months post-transplantation so we could examine tissue before the onset of reactive astro- and microgliosis (2 weeks), at the early stages of reactive gliosis (4 weeks) and at a chronic stage of gliosis (3 months) based on data from our previous work in this model (Martins et al., 2001; Bates et al., 2007).

2.1 Evans blue dye injection and immunohistochemistry

At 2 weeks, 4 weeks and 3 months post-transplantation, animals were injected i.p. with EBD (1% w/v in PBS, 100μl/10 g body weight) 24 hours before sacrifice with sodium pentobarbitone (Hamer et al., 2002).

Brains were removed and slowly frozen in iso-pentane. Unfixed, frozen sections (10μm) were collected on gelatin-coated slides and stored at –20°C until EBD visualization and immunohistochemical staining. Frozen sections were removed from –20°C and thawed at room temperature for 5 minutes. The relationship between the basement membrane of blood vessels and activated astrocytes was examined using laminin (rabbit anti-mouse, catalogue number 10765, AB_1542225, 1:200 dilution, ICN/American Research Products USA) and GFAP (monoclonal anti-mouse clone G-A-5, catalogue number G3893, AB_477010, 1:400 dilution, Sigma-Aldrich USA) immunohistochemistry. Blood vessels (endothelial cells) were immunostained using PECAM-1 (CD-31) reactivity (monoclonal anti-mouse, catalogue number CBL1337, AB_2283583, 1:100 dilution, Merck Millipore USA) and percentage vascularity determined using the method described by Smythe and colleagues (Smythe et al., 2002). The immunostaining protocol followed that described previously (Martins et al., 2001; Bates et al., 2007), except that for PECAM-1 detection, sections were fixed in ice-cold methanol for 1 minute. For all other antibodies, sections were fixed in 4% paraformaldehyde for 5 minutes. Note that homotopic cortex-to-cortex grafts were well-integrated into host cortex, thus in the fresh, frozen tissue sections needed for EBD analysis, grafted tissue was not always easy to discriminate from adjacent host tissue.

Images were taken using a CoolSnap camera attached to a Nikon Eclipse E800 microscope and composite images of PECAM-1 staining were obtained using a LEICA DM RB/E microscope with XYZ motorized stage. The area of the grafted tissue was calculated using Image-Pro Plus (Version 4) software.

The brightness of EBD staining within the cortex, graft and interface between the host and grafted tissue was analyzed using NIH Image (ImageJ 64, NIH). A M158 Weibel grid was overlaid over the images and the number of point-vessel intersections (i.e. within a blood vessel lumen or within the blood vessel wall) counted in order to measure the vascularity of the tissue. At least 3 frames were used for each region of interest (i.e. graft, adjacent cortex, contralateral cortex) on each tissue section. The formula for percentage vascularity was given by % vascularity (sample) = [P_C/(F×P_T)] ×100 where PC = total number of points falling on a vascular space (combined for all field counts), F = total number of field counts for each sample and PT = total number of points on the grid (i.e. 168). The average percentage vascularity was then calculated.

2.2 ELISA for protein measurement of IL1β, IL6 and TNFα

Animals from 2 week, 4 week and 3 month post-transplantation time-points were used for ELISA. Host cortex, homotopic grafts, heterotopic grafts and host midbrain tissue was extracted and immediately frozen in liquid nitrogen for storage at –80°C. Protein was extracted from the tissue via homogenization with RIPA buffer (150 mM NaCl, 50 mM Tris HCl pH 7.5, 1% triton X-100, 0.5% sodium deoxycholate, 0.1% SDS and 0.1 mM PMSF). Protein concentration was determined using the Micro BCA protein assay kit (Pierce, IL USA) as detailed in (Bates et al., 2002, 2014).

The concentration of IL1β, IL6 and TNFα was measured using Quantikine^® kits (R&D Systems Inc., MN, USA). In order to achieve the best compromise between tissue availability and validity of ELISA data, each ELISA was optimized for protein concentration, resulting in 50μg total protein loaded for IL1β and 200μg for IL6 and TNFα The optical density of each well was then measured using a microplate reader set to 450 nm. This reading was adjusted by a correction reading taken at 540 nm. All samples were tested in duplicate. Positive controls of a known concentration (supplied in each kit) and negative controls containing only assay diluent (blank) were used to ensure specific results. The data were adjusted to 200 pg of total protein to allow for comparison between cytokines (expressed as pg/200μg).

All statistical analyses were performed using one-way ANOVA at the 0.05 significance level using GraphPad Prism (version 4). For inflammation and vascularity studies, parametric data were analyzed using ANOVA with Bonferroni post-hoc analysis. For EBD quantification and elsewhere were data were not normally distributed, data were analyzed using the Kruskal Wallis method with Dunns post-hoc analysis.

3 Results

3.1 Blood brain barrier integrity and vascularization of cortex transplants

We were unable to distinguish any obvious differences in either EBD staining or immunohistochemical staining in homotopic cortex grafts relative to adjacent host cortical tissue. However, heterotopic transplants examined 2 weeks, 4 weeks and 3 months post-transplantation displayed extensive extravasation of EBD at the host-graft interface at all time-points studied (Fig. 1A-C). There was minimal EBD extravasation from blood vessels within heterotopic cortex-to-midbrain grafts, and the degree of EBD extravasation was higher at the host-graft interface at the 2 week, 4 week and 3 month time-points, compared to host cortical areas (Fig. 1D, p≤0.05). These data indicate that heterotopic transplantation results in a loss of BBB integrity that is already evident at 2 weeks, particularly close to the host-graft interface.

The relationship of reactive astrocytes to blood vessels within grafted tissue was studied using double immunofluorescence for GFAP and laminin reactivity. At 2 weeks post-transplantation, we observed vessels traversing the graft-host interface (Fig. 2A, D, G). Coupled with the presence of EBD within the graft itself, we deduced that the graft vasculature was continuous with that of the host, in agreement with previous transplantation studies (Lund et al. 1981; Rosenstein 2000) indicating that blood vessels from the host grew into the grafted tissue.

Minimal GFAP reactivity was observed at the host-graft interface at 2 weeks post transplantation (Fig. 2B). In contrast, by 4 weeks post transplantation, extensive GFAP reactivity was observed within heterotopic grafts (Fig. 2E), consistent with an earlier immunohistochemical time-course study (Bates et al., 2007). Importantly however, blood vessels within these grafts had an abnormal shape, appearing to be of wider diameter with uneven walls compared to vessels from the underlying host tissue (arrows, Fig. 2F). Increased GFAP reactivity co-localized with laminin staining was observed around these abnormal vessels (Fig. 2F). Intense GFAP staining and abnormal vascular profiles continued to be a characteristic feature of these grafts 3 months post transplantation (Fig. 2G-I).

The extent of vascularization in heterotopic grafts compared to normal host tissue was quantified using PECAM-1 immunohistochemistry. The number of PECAM-1 positive vascular profiles was notably lower in 2-week old grafts compared to the surrounding cortex and adjacent areas (Fig. 3A). As the grafts aged, this imbalance between PECAM-1 labeled vessels in graft versus host tissue appeared to resolve and reactivity within grafts became similar to surrounding host tissue (Fig. 3B, C). Quantitative analysis of graft tissue confirmed this developmental pattern. Percentage vascularity in 2 week-old grafts was significantly lower compared to normal host tissue and grafts at later time points (p < 0.05, Fig. 3C). As the grafts aged, the vascularity of grafts increased to be comparable to that of host tissue. A similar pattern was observed when percentage vascularity was calculated for laminin-stained sections (Fig. 3D). This occurred despite continued permeability of the BBB at the host-graft interface.

3.2 Inflammatory changes in homotopic versus. heterotopic grafts

3.2.1 ELISA for IL1β, IL6 and TNFα

Because different amounts of total protein were assayed for the different cytokines, results have been normalized to 200 pg of total protein so that the levels of each of the cytokines can be compared. The protein concentration for IL1β (Fig. 4A) was constant for the control host cortex throughout all of the time points, consistent with previous studies (Lechan et al., 1990). When comparing protein expression of IL1β in the heterotopic grafts at the different time points, there was a significant increase in IL1β at 4 weeks (p < 0.01) that persisted at 3 months (p < 0.05), compared to 2 weeks. IL1β protein levels in homotopic grafts were significantly higher than in heterotopic grafts at 2 weeks (p < 0.05, Bonferroni). This expression reduced with time until it was similar to control levels at 3 months. There was no significant difference in IL6 expression across the time points for any type of tissue (Fig. 4B).

We conducted optimization experiments and concluded that 200μg of protein was required for TNFα ELISA. Heterotopic grafts provided sufficient material for analysis but we were unable to test the homotopic graft samples. There was minimal TNFα in the host brain and in heterotopic grafts at 2 and 4 weeks, but a strong trend suggesting that TNFα levels were increased in heterotopic grafts at 3 months (p = 0.054, Fig. 4C).

4 Discussion

We have shown that BBB leakage and vascular insufficiency is evident at 2 weeks post-transplantation. This occurred prior to both gliotic and inflammatory/cytokine changes that became evident at 4 weeks, and likely before enhanced CSPG deposition, known to be present by 3 months (Harvey et al., 1997). Together, the results suggest that exposure of heterotopic cortical grafts to substances normally excluded by the BBB and/or ischemia-like conditions may be a significant contributing factor to establishing gliosis in this model, and that inflammation alone was not responsible for initiating gliosis.

The timing of cytokine up-regulation suggests that cytokine production was a consequence, rather than a cause, of gliosis. Inflammatory cytokines may in turn contribute to the oxidative stress (Bates et al., 2007) and altered APP processing (Martins et al., 2001; Bates et al., 2002) previously documented in heterotopic cortex grafts. The latter data lend support to the idea that chronic gliosis can accelerate AD-like pathology. Anti-inflammatory treatment can ameliorate oxidative stress, with no effect on astrocyte activation, lending additional support to our conclusion that inflammation is a secondary, rather than primary event in this model (Bates et al., 2007).

By its nature, transplantation of neural material is likely to induce breakdown of the BBB. For example, intraventricular transplantation leads to increased BBB permeability (Rosenstein, 1987) and marked astrogliosis in close proximity to graft vasculature (Krum & Rosenstein, 1989; Rosenstein, 1987). In our model, we were unable to detect any changes in homotopic grafts in tissue sections, despite careful analysis of serial sections in our vasculature studies. This suggests that the changes we observed in terms of BBB permeability and vasculature were not due to the transplantation procedure per se. Previous studies suggest that homotopic transplants of embryonic tissue integrate functionally and anatomically with the host cortex (Jaeger et al., 1980, 1981; Lund et al., 1981; Harvey et al., 1997; Martins et al., 2001; Bates et al., 2002; Gaillard et al., 2007) consistent with the lack of BBB permeability and normal vascularization in this type ofgraft.

In our study, we observed that reactive astrocytes surrounded the blood vessels within gliotic grafts, suggesting that alteration to BBB permeability can result in astrogliosis and associated pathological changes. Interestingly, the astrocytic scar-like barriers around cerebral blood vessels have been shown to prevent the influx of leukocytes and protect CNS function in experimental autoimmune encephalitis (EAE) (Voskuhl et al., 2009), highlighting the context-dependent consequences of reactive gliosis. BBB dysfunction is acknowledged as an important component to the pathological cascade of AD (Deane & Zlokovic, 2007).

Neuroscientists are discovering numerous brain-region specific differences and indeed astrocytes display regional heterogeneity (Tabata, 2015; Tsai et al., 2012). Furthermore, astrocytes are able to sense and react to highly localized cellular environments in response to brain injury (Burda et al., 2015). Previous studies have shown that heterotopic transplantation of fetal midbrain (tectum) to cortex does not elicit a massive increase in glial reactivity in the grafted tissue (Tan & Harvey, 1999). Midbrain-to-midbrain transplantation also leads to a low level astrocytic response (Harvey, 1994; Harvey, Plant, & Kent, 1993), but the extent of this response is minor compared to the astrocytic reactivity seen in cortex-to-midbrain grafts. In addition to regional-specificity in terms of astrocyte biology, there are also regional-specific differences in response to injury and insult. Therefore, one possibility for why chronic gliosis occurs in heterotopic transplantation to the midbrain is that there is a tissue mis-match in metabolic requirements between midbrain and cortically derived donor material. Recent brain-imaging data have begun to elucidate brain-region specific metabolic requirements and show cerebral cortex is more metabolically active than midbrain structures such as the superior colliculus in healthy human subjects (Heiss et al., 2004).

Chronic hypoxia results in regional-specific changes in angiogenesis, with cerebrum, hippocampus and striatum upregulating angiogenic sprouting, compared to moderate angiogenesis in the cerebellum and medulla (Patt, Sampaolo, Theallier-Janko, Tschairkin, & Cervos-Navarro, 1997). Astrocytes promote angiogenesis in the developing cortex (Ma, Kwon, & Huang, 2012) and in the retina (Gariano & Gardner, 2005), and the interaction between blood vessels and glial progenitors has been shown to influence astrocyte differentiation in the postnatal period (Zerlin & Goldman, 1997). Vascular endothelial growth factor (VEGF) is strongly expressed by astrocytes following injury (Krum & Rosenstein, 1998) and under ischemic conditions via the hypoxia-inducible factor (HIF) family of transcription factors (Bergeron, Yu, Solway, Semenza, & Sharp, 1999). It is therefore possible that heterotopic transplantation disrupts VEGF/angiogenic signaling resulting in abnormal astrocyte function. Consequently, our model can be used in future studies to further examine the relationship between chronic gliosis and angiogenesis, including the role of VEGF and/or identify other molecular players in this process (Buffo, Rolando, & Ceruti, 2010; Lee, Han, Bai, & Kim, 2009; Rosenstein & Krum, 2004).

In summary, the fetal transplantation model has potential as a system for further elucidation of the relationship between reactive gliosis, regional-specific cell variability and angiogenesis. It is likely that reduced vascular density at 2 weeks post transplantation resulted in a degree of hypoxia in heterotopic grafts that contributed to the long-term, chronic glial pathology observed in this model, including inflammation. The integrity of the BBB at the host-graft interface was disrupted throughout the time course studied, which in turn may allow the exudation of a number of blood-borne substances into the normally protected neuropil environment. This transplantation model may therefore be able to address some of the complex interrelationships between vascular insufficiency, gliosis, astrocyte dysfunction and neuropathology in terms of both neurodegenerative disease processes and therapeutic strategies involving transplantation and regenerative medicine (Sekiya et al., 2015).

Footnotes

ACKNOWLEDGMENTS

The authors acknowledge Ms Margaret Pollett and Ms Natalie Symons for technical support and Ms Marissa Penrose for help with the figures.

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