Modulation of post-stroke degenerative and regenerative processes and subacute protection by site-targeted inhibition of the alternative pathway of complement
- Ali Alawieh†1,
- Andrew Elvington†1,
- Hong Zhu2,
- Jin Yu2,
- Mark S. Kindy2, 3,
- Carl Atkinson1 and
- Stephen Tomlinson1, 3Email author
© Alawieh et al. 2015
Received: 8 October 2015
Accepted: 20 December 2015
Published: 30 December 2015
Complement promotes neuroinflammation and injury in models of stroke. However, complement is also being increasingly implicated in repair and regeneration after central nervous system (CNS) injury, and some complement deficiencies have been shown to provide acute, but not subacute, protection after murine stroke. Here, we investigate the dual role of complement in injury and repair after cerebral ischemia and reperfusion.
We used complement-deficient mice and different complement inhibitors in a model of transient middle cerebral artery occlusion to investigate complement-dependent cellular and molecular changes that occur through the subacute phase after stroke.
C3 deficiency and site-targeted complement inhibition with either CR2-Crry (inhibits all pathways) or CR2-fH (inhibits alternative pathway) significantly reduced infarct size, reduced apoptotic cell death, and improved neurological deficit score in the acute phase after stroke. However, only in CR2-fH-treated mice was there sustained protection with no evolution of injury in the subacute phase. Whereas both inhibitors significantly reduced microglia/macrophage activation and astrogliosis in the subacute phase, only CR2-fH improved neurological deficit and locomotor function, maintained neurogenesis markers, enhanced neuronal migration, and increased VEGF expression. These findings in CR2-fH-treated mice correlated with improved performance in spatial learning and passive avoidance tasks. The complement anaphylatoxins have been implicated in repair and regenerative mechanisms after CNS injury, and in this context CR2-fH significantly reduced, but did not eliminate the generation of C5a within the brain, unlike CR2-Crry that completely blocked C5a generation. Gene expression profiling revealed that CR2-fH treatment downregulated genes associated with apoptosis, TGFβ signaling, and neutrophil activation, and decreased neutrophil infiltration was confirmed by immunohistochemistry. CR2-fH upregulated genes for neural growth factor and mediators of neurogenesis and neuronal migration. Live animal imaging demonstrated that following intravenous injection, CR2-fH targeted specifically to the post-ischemic brain, with a tissue half-life of 48.5 h. Finally, unlike C3 deficiency, targeted complement inhibition did not increase susceptibility to lethal post-stroke infection, an important consideration for stroke patients.
Ischemic brain tissue-targeted and selective inhibition of alternative complement pathway provide self-limiting inhibition of complement activation and reduces acute injury while maintaining complement-dependent recovery mechanisms into the subacute phase after stroke.
Following onset of cerebral ischemia, many stroke patients show reperfusion of their infarct either spontaneously or as a secondary effect of thrombolytic therapy. Cerebral reperfusion initiates a cascade of pathophysiological events that cause secondary injury, which can lead to greater tissue damage and more severe functional and cognitive deficits. Clinical observations and experimental studies indicate a central role for complement in the propagation of ischemia reperfusion injury (IRI) in both the central nervous system (CNS) and in non-CNS tissue [1, 2].
Cumulative evidence indicates that following cerebral ischemia and reperfusion, complement is activated via the lectin pathway and amplified via the alternative pathway (reviewed in ). Deficiency or pharmacologic inhibition of either pathway is protective in the acute phase following murine ischemic stroke [4–8]. However, from a clinical standpoint, it is important to determine the effect of any potential therapeutic strategy on longer-term outcome after stroke. While complement activation may be injurious in the acute phase, there is evidence that complement also has neuroprotective functions in the CNS, including after stroke [9, 10]. In this regard, it has been shown that the protective effect of lectin pathway deficiency is not sustained in the subacute phase of stroke , even though there is a wide therapeutic window for lectin pathway inhibition and acute protection . In addition, deficiency in C3, a central protein of all complement pathways, provides effective protection in the acute phase [12, 13], but not the subacute phase following ischemic stroke .
In a previous study, we demonstrated that alternative pathway-deficient (fB−/−) and inhibited (CR2-fH treated)-mice are protected from acute injury after cerebral ischemia and reperfusion. We further demonstrated that alternative pathway inhibition resulted in reduced infarct volumes and reduced neurological deficit scores in the subacute phase (7 days) after ischemia . In the current study, we follow up on this preliminary observation and investigate how complement modulates the cellular and molecular events that contribute to post-stroke degenerative and regenerative processes, and how this relates to cognitive impairment and functional recovery. We also investigate whether subacute protection is a specific property of the alternative pathway. For this study, we utilize CR2-fH, an inhibitor shown to specifically inhibit the alternative pathway , and CR2-Crry, an inhibitor of all complement pathways that has been shown to provide acute protection after stroke, but that has not been investigated with regard to subacute outcome [13, 16]. The complement inhibitors Crry and fH are targeted to sites of complement activation (C3d deposition) via the complement receptor 2 (CR2) moiety .
CR2 fusion proteins
The complement inhibitors CR2-Crry and CR2-fH were constructed, expressed, and purified as previously described [15, 16]. For quality control, complement inhibitory activity was tested by Zymosan assay as described .
Transient ischemic stroke model
Two cohorts of male 8- to 10-week-old wt C57BL/6 mice or C3−/− mice on C57BL/6 background (Jackson Laboratories) were independently subjected to transient ischemic stroke, with 60-min middle cerebral artery occlusion (MCAO) . In the first cohort, wt mice received one treatment of 0.25 mg CR2-Crry, 0.4 mg CR2-fH, or 100 μl phosphate buffered saline (PBS) vehicle given intravenously 30 min following reperfusion. Dosing was based on efficacy titrations performed in a model of intestinal IRI [15, 16]. Following either 24 h or 7 days of reperfusion, animals were sacrificed and brains processed for histology or protein extraction. For antibiotic prophylaxis, C3−/− mice received 250 mg/L ciprofloxacin (VetSource) in drinking water ad libitum 14 days prior to MCAO and until sacrifice. In the second cohort, wt mice received either one treatment of 0.4 mg CR2-fH or 100 μl PBS vehicle 30 min following reperfusion. Following either 3 or 7 days of reperfusion, mice were assessed for neurological outcome as well as performance on Barnes maze and passive avoidance tests. Mice were sacrificed on day 7 and brains processed for histology or protein extraction. Cerebral blood flow was assessed by Laser Doppler Flowmetry (Moor Instruments) before, during, and following ischemia; mice were excluded if not achieving a reduction in blood flow to 20 % of pre-ischemia levels. Temperature, blood pressure, and heart rate were also monitored before, during, and following ischemia, and consistent with previous reports, no differences were observed . All groups were randomly assigned, and experimenters were blinded to identity of groups. All experiments and procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals by the National Institutes of Health and approved by the Institutional Animal Care and Use Committee at the Medical University of South Carolina.
Neurological deficit was assessed following MCAO by a blinded observer using a five-point scoring system, as previously described . Additionally, locomotor activity was automatically quantified using the Versamax open field activity monitor (AccuScan Instruments). Mice were placed in a random corner and allowed to acclimate for 10 min prior to a 60-min testing period. External noise, lights, and other stimuli were minimized to reduce bias. Several measures were automatically retrieved during the task including total distance moved, number of movements, time spent at periphery, and time spent at the center. Activity readings taken prior to sham procedure were used to establish any differences in baseline activities. The duration that the animal spent at the periphery vs. the center was used to assess anxiety level during the task.
Animals of the second cohort were tested for their performance on both Barnes maze and passive avoidance tasks. To assess spatial reference memory, mice were trained on Barnes maze for 5 days before surgery, as previously described , then tested again on days 3 and 7 after reperfusion for the time needed to escape into the hole, the number of error pokes, and the length of the animal’s path prior to escape. An automated passive avoidance apparatus (Coulbourn Instruments) was used to assess avoidance learning with automated sensing and shock systems (GraphicState® 4, Coulbourn Instruments). The apparatus included a double compartment chamber with one lit and one dark compartment. Mice were allowed to explore the chamber for 5 min on habituation phase. Following habituation, the mice were given one trial where a shock is associated with the dark side, allowed 48 h of rest, and then tested for retention measured as latency to enter the dark side. Testing was repeated on days 3 and 7 post-reperfusion with no shock delivered during test phase.
Histological and immunohistochemical analysis
For analysis of infarct volume, brains were perfused transcardially with PBS before their removal, then cut into 2-mm coronal sections and stained with 2 % triphenyltetrazolium chloride (TTC) . Infarct volumes were analyzed with ImageJ software (National Institutes of Health) and calculated as percentage of total brain from summation of four sections obtained 2 mm apart. Immunohistochemical staining was conducted on 8-μm paraffin sections and assessed by a blinded observer by light microscopy (Olympus BX61). Following antigen retrieval (IHC World), the following primary antibodies were used: anti-ionized calcium-binding adaptor molecule 1 (Iba-1, 1:250; Abcam), anti-mouse glial fibrillary acidic protein (GFAP, 1:1000; Dako), anti-von Willibrand Factor (vWF, 1:500; Dako), anti-Ki67 (1:500; Abcam), anti-doublecortin (Dcx, 1:200; Millipore), and anti-Gr-1 (1:100, Stem Cell). Primary antibodies were detected with ImmPress-HRP kit and NovaRed peroxidase chromagen (Vector Laboratories), and primary antibodies were omitted for negative controls. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was performed using ApopTag Peroxidase Staining Kit (Millipore) per manufacturer’s instructions. For birth dating proliferating neuroblasts, intraperitoneal BrdU was administered to a subset of mice every other day starting 24 h after reperfusion, and BrdU-positive cells were detected by anti-BrdU antibody (1:300; Sigma).
Quantification of staining
Paraffin sections were obtained +2 to −2 mm relative to bregma, and sections 200 μm apart were used for quantification. The ischemic core and the penumbra (area surrounding the ischemic core) were confirmed with Luxol fast blue staining or counterstaining, and random fields were examined within the penumbra using an automated motorized stage on an Olympus BX61 using Visiopharm software. Counting of apoptotic cells was performed on an average of eight sections per brain, with the entire ipsilateral hemisphere being quantified, and density was calculated as number of positive cells per square millimeter of tissue. Reactive astrocytes, microglia/macrophages, and vWF positive vessels were quantified on sections 200 μm apart. An average of five sections/brain was quantified. Cells were counted in randomly generated fields, and density was calculated as number of positive cells per square millimeter of tissue. Dcx- and Ki67-positive cells were quantified within the subventricular zone (SVZ) of the lateral ventricles, the subgranular zone (SGZ) of the dentate gyrus, the basal ganglia, and the hippocampus from ×40 magnification random fields on sections 25 to 50 μm apart (three sections/brain). All analyses were done by light microscopy (Olympus BX61 with Visiopharm image acquisition software) by an observer blinded to the experimental groups.
Tissue protein analysis
Following sacrifice, brains were divided into ipsilateral and contralateral hemispheres, and protein was extracted by homogenization in NP-40 lysis buffer (Invitrogen) containing 1 mm PMSF (Sigma-Aldrich), 92.6 μm FUT175 (BD Biosciences) and 5 μL of protease inhibitor cocktail (Sigma-Aldrich). Protein concentrations were determined using BCA protein assay kit (Thermo Scientific). Vascular endothelial growth factor (VEGF; Abcam), complement C5a (BD Biosciences), and β-actin (Sigma-Aldrich) were assessed through Western blotting of 25 μg total protein, with SDS-PAGE run under reducing conditions, and detected with HRP-conjugated secondary antibodies (Vector Laboratories). Signal densities were calculated relative to β-actin and normalized to wt groups.
Eight C57BL/6 mice (8 weeks of age) were also subjected to 1 h MCAO and treated with either CR2-fH or PBS vehicle (n = 4/group) to assess the effect of CR2-fH on gene expression profile after MCAO. Animals were perfused with PBS 72 h after reperfusion, and their brains were extracted for RNA Extraction. Gene expression analysis was performed using the Nanostring nCounter Analysis System (Nanostring Technologies) using a custom-designed codeset containing 249 genes involved in immunological and cell survival processes (Additional file 1) . Each reaction contained 250 ng of total RNA in a 5-μL aliquot, plus reporter and capture probes, and six pairs of positive control and eight pairs of negative control probes. Analysis and normalization of the raw Nanostring data was conducted using nSolver Analysis Software v1.1 (Nanostring Technologies). Raw counts were normalized to levels of reference gene. A background count level was estimated using the average count of the eight negative control probes in every reaction plus two SDs.
Five C57BL/6 mice (8 weeks of age) had their heads shaved and treated with Nair 2 days prior to MCAO surgery described above. Fluorescently labeled CR2-fH was injected according to the therapeutic protocol described above. CR2-fH was labeled using Xenolight C750 NIR Fluorescent Dye according to manufacturers instructions (Perkin Elmer). Mice were anesthetized and imaged at 6, 24, 48, 72 and 96 h and at 7 days post-injection using a Maestro EX imaging system (Perkin Elmer). Sham-operated animals were also injected as controls. Fluorescent signal was quantified with supplied software, and tissue half-life was calculated according to the formula t 1/2 = t × ln(2)/ln(N 0/N t), where t = fluorescence signal after time gone by, N 0 = signal at beginning (6 h), N t = signal after period of time (90 h).
Statistical differences between parametric data (infarct volumes, activity values, ELISA values, cell counts, densitometry, and Nanostring data) were assessed using one-way analysis of variance (ANOVA) test with Bonferroni’s multi-group comparison, and non-parametric data (neurological deficits) were compared with the Kruskal-Wallis test with Dunn’s comparison (Prism 5.0, GraphPad). Survival was compared from all mice subjected to MCAO using the Kaplan-Meier test. Differences between data were considered statistically significant when p < 0.05.
Targeted complement inhibition reduces the extent of injury post-MCAO
Targeted complement inhibition reduces subacute microglia/macrophage activation and astrogliosis after MCAO
Effect of complement inhibition on neurological outcome and locomotor function after MCAO
To more objectively assess neurological deficit, we measured locomotor activity using an open field activity monitor. There was no significant difference in locomotor activity between any group up to 3 days post-MCAO, but on day 7, CR2-fH-treated mice had significantly increased locomotor activity compared to all other groups (Fig. 3b, c). Since a difference in anxiety levels may confound performance on open field as well as other tasks, we also assessed anxiety levels across the groups by measuring the percentage of time spent at the center of an open field. We found no significant differences in anxiety levels among the different groups on days 3 and 7 post-MCAO (Fig. 3d). The picture emerging from the data thus far indicates that in the subacute phase after stroke, CR2-fH provides better protection than CR2-Crry or C3−/− in terms of evolution of secondary injury and neurological outcome.
CR2-fH maintains neurogenesis and enhances neuronal migration in the subacute phase after MCAO
CR2-fH less effectively blocks complement activation than CR2-Crry
Profiling of gene expression changes after CR2-fH treatment.
CR2-fH increases subacute VEGF levels in the brain after MCAO
CR2-fH improves spatial reference memory and avoidance learning in the subacute phase after stroke
On Barnes Maze task 3 days post-MCAO, the performance of wt control mice deteriorated significantly, as manifested by an increase in latency to escape, path length, and number of error pokes compared to pre-surgical baseline. By day 7 post-MCAO, performance of this group was improved, but was still significantly worse compared to pre-surgical baseline (p < 0.05). In contrast, CR2-fH-treated mice displayed no impairment on Barnes maze performance at either 3 or 7 days post-MCAO compared to pre-surgical baseline (Fig. 9b–d). Thus, compared to wt controls, CR2-fH treatment significantly improved spatial reference memory through the subacute phase after ischemic stroke.
To control for possible cognitive-enhancing effects of CR2-fH, we also treated sham-operated mice with CR2-fH and tested cognitive performance after sham surgery on both tasks. CR2-fH treatment did not enhance cognitive performance in sham-operated mice (Fig. 9). The above data together show that a single post-reperfusion dose of CR2-fH not only improves neurological/locomotor and histological measures in the subacute phase after stroke, but also improves cognitive performance.
CR2-fH treatment reduces neutrophil infiltration into the brain after MCAO
Acutely administered CR2-fH targets to the post-ischemic brain and persists into the subacute phase after stroke
Complement inhibition does not increase post-stroke mortality
Complement-mediated inflammation has been shown to play an important role in the progressive degenerative events that take place after ischemic stroke, but there is increasing evidence that complement is also involved in subsequent repair and regenerative mechanisms that occur during recovery . Numerous studies have shown that complement deficiency or inhibition is protective in the acute phase after stroke (24–48 h), but it has also been shown that complement deficiency can worsen subacute outcomes [11, 14]. The reasons for the differences in acute and subacute outcomes are not well understood, but it is clear that complement has a balancing role in injury vs. protection in many pathological conditions, and differences in acute and subacute outcomes after stroke may be due to balancing roles for complement in early inflammation and injury vs. subsequent neuroprotection and neurogenesis. In the context of neuroprotective functions of complement, both C3a and C5a have been shown to be protective against excitotoxic neuronal injury [30–32], and C3a has a protective role in neonatal hypoxia-ischemia brain injury , even though C1q exacerbates injury in a similar model . C3a is also implicated in promoting neurogenesis, both basal  and following ischemic stroke , and similar to C5a, it has been shown to regulate the differentiation and migration of neural progenitor cells in vitro [35, 36]. In addition, complement opsonins of the classical (C1q), lectin (MBL, ficolins), and alternative (properdin) pathways, as well as C3 opsonins common to all pathways, promote the clearance of apoptotic cells and debris, which is important for the resolution of inflammation and recovery [35, 37, 38].
The use of two different complement inhibitors in this study allowed us to investigate the selective contribution of the alternative complement pathway in secondary injury after stroke. Of the two complement inhibitors investigated, only CR2-fH improved neurological deficit and locomotor function at 7 days after stroke. Improved motor outcome at 7 days in CR2-fH-treated mice compared to CR2-Crry-treated mice and C3-deficient mice is associated with significantly lower cortical cell death in the CR2-fH-treated group. In addition, only CR2-fH-treated mice had significantly reduced hippocampal cell death, and only CR2-fH-treated mice did not display a reduction in neurogenesis markers in the SVZ. In addition to maintaining post-stroke neurogenesis, CR2-fH treatment also promoted significantly more neuroblast migration from the SVG compared to wt mice, findings that correlated with improved performance on spatial learning and passive avoidance tasks in the subacute phase after stroke. Since complement activation products are implicated in acute injury as well as recovery and neurogenesis after CNS injury, a mechanism accounting for the different subacute outcomes in CR2-fH- vs. CR2-Crry-treated mice may be related to a difference in the extent of complement activation. Current evidence indicates that following cerebral ischemia and reperfusion, complement is activated by the lectin pathway [5–9] and amplified by the alternative pathway . As outlined above, C3a and C5a have neuroprotective/regenerative roles, and whereas CR2-fH treatment significantly inhibited C5a generation in the ipsilateral brain for up to 3 days after stroke, C5a levels were still significantly higher than in brains from CR2-Crry-treated mice. Thus, CR2-fH maintains a baseline level of C5a generation through the subacute phase that correlates with improved overall post-stroke recovery, and based on previous data, this likely occurs via the lectin pathway. Differences in the extent of complement activation could also account for our observations that CR2-Crry is more effective than CR2-fH at reducing acute injury (lesion size), even though longer-term outcomes are worse. The concept of CR2-fH providing optimal longer-term protection by self-limited complement activation is further supported by findings that high-dose, but not low-dose, C3aR antagonism impaired post-stroke SVZ neurogenesis in the subacute phase [14, 24]. CR2-fH, but not CR2-Crry, also increased VEGF expression within the ipsilateral hemisphere, and this may point to another protective mechanism of CR2-fH. Although the role of complement in post-stroke angiogenesis has not been investigated, C3a and C5a have been shown to promote VEGF expression and pathogenic neovascularization within the retina in a model of age-related macular degeneration . We did not observe differences in angiogenesis with complement inhibition, but it is possible that this may occur at later time points.
Regarding post-stroke neurogenesis, findings are mixed on whether newly forming neurons make a functional impact on plasticity and recovery. Here, we investigated neurogenesis and neuroblast migration 7 days following ischemic stroke, and show that increased neuroblast migration to the basal ganglia and hippocampal area in CR2-fH-treated mice is indeed associated with improved performance on cognitive and memory tasks. The effect of CR2-fH on neurogenesis and neuroblast migration was also associated with increased levels of NCAM and VEGF transcript and VEGF protein. Both molecules have been studied in the context of neurodevelopment, with VEGF expression shown to induce neuroblast proliferation and migration, while NCAM is a neuronal adhesion molecule essential for the formation of chains of migrating neuroblasts during development  It is noteworthy that CR2 expression in neural progenitor cells has been shown to regulate hippocampal neurogenesis, and treatment with the CR2 ligand, C3d, was shown to reduce the number of proliferating neuroblasts in vivo . It is therefore possible that the CR2 targeting moiety of CR2-fH could be contributing to its function by competing with the interaction of C3d with CR2 expressed on neural progenitor cells, although it is unlikely this represents a principle mechanism of action since there is inhibition of neurogenesis in CR2-Crry-treated mice compared to wt.
The C3a and C5a anaphylatoxins are implicated in promoting immune cell migration and infiltration, and may also be involved in neuroblast migration. Interestingly, however, while we show that the significant reduction of C5a levels in the brains of CR2-fH-treated mice is associated with decreased immune cell infiltration (especially neutrophils), it is associated with enhanced neuroblast migration. CR2-fH inhibited but did not completely block C5a generation, and there may be different thresholds for anaphylatoxin stimulation or different mechanisms of action of the anaphylatoxins in immune cell vs. neuroblast migration, and these data again highlight the dual role of complement in injury and repair.
Additional differences between C3-deficient and complement-inhibited mice were the extent of microglial activation and post-stroke mortality. C3-deficient mice had significantly higher activation of microglial/macrophage cells compared to complement-inhibited mice. The reason for the higher levels of microglial/macrophage activation in C3-deficient mice is not clear, but may be related to the increased levels of striatal apoptosis found in complement-deficient vs. inhibited mice. In this regard, C3 opsonization of apoptotic cells is known to play a role in apoptotic cell clearance, and the complete absence of this process in C3-deficient mice may more severely restrict cell clearance and impair the resolution of inflammation. In addition, C3-deficient mice had a significantly higher mortality rate compared to complement-inhibited mice. Treatment of C3-deficient mice with an antibiotic eliminated this difference, indicating that increased mortality was due to infection. Complement plays an important role in host defense mechanisms, and our finding here is also in accord with a previous study in which it was shown that C3 deficiency, but not CR2-Crry treatment, increased susceptibility to infection in a model of septic peritonitis . CR2-mediated targeting obviates the need for systemic complement inhibition [15, 16], and the current data indicate that CR2-fH does not increase susceptibility to infection following experimental ischemic stroke. Secondary infectious complications are a serious concern for stroke patients, and infection (septicemia and pneumonia) is a major cause of death following stroke in the model we use here . Other known causes of mortality after stroke include hemorrhagic transformation, brain edema and, with regard to experimental stroke, loss of mobility leading to malnutrition and dehydration of the animal.
In conclusion, we show that an acutely administered dose of CR2-fH reduced injury and improved neurological and behavioral outcomes in the subacute phase after stroke. These improved outcomes were linked to reduced extent of cell death and increased neurogenesis and VEGF expression. The improved longer-term outcomes in CR2-fH-treated mice compared to CR2-Crry-treated and C3-deficient mice, and the inhibition of neurogenesis in CR2-Crry-treated and C3-deficient mice, indicate that CR2-fH provides targeted and self-limiting complement inhibition that dissects the dual role of complement in injury and recovery after stroke. This conclusion is supported by data showing that CR2-Crry more completely blocks complement activation than CR2-fH, as determined by comparing post-stroke brain levels of the complement activation product, C5a. This approach of alternative pathway inhibition offers potential advantages over systemic and/or regulated doses of complement inhibitors that may be contraindicative to long-term outcome. The current findings also warrant further investigation into therapeutically relevant issues such post-stroke therapeutic window, outcomes in older mice with increased stroke risk factors or with pre-existing cerebrovascular disease, and outcomes in pre- and post-menopausal female mice.
This work was supported by grants from the NIH (1P20GM109040), the Department of Veterans Affairs (Merit Award 1I01RX001141 and 1BX001218), and an American Heart Association Pre-doctoral Fellowship to AA (15PRE25250009).
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