NogoA Neutralization Promotes Axonal Restoration After White Matter Injury In Subcortical Stroke

Blocking axonal growth inhibitor NogoA has been of great interest for promoting axonal recovery from neurological diseases. The present study investigates the therapeutic effects of blocking NogoA, inducing functional recovery and promoting white matter repair in an experimental animal model of stroke. Adult male rats were subjected to white matter injury by subcortical ischemic stroke. Twenty-four hours after surgery, 250 ug of anti-NogoA or anti-IgG-1 were administered through the tail vein. The quantity of NogoA protein was determined by immunohistochemistry in the brain and peripheral organs. In addition, functional status, lesion size, fiber tract integrity, axonal sprouting and white matter repair markers were analyzed. Moreover, an in vitro study was performed in order to strengthen the results obtained in vivo. A lower quantity of NogoA protein was found in the brain and peripheral organs of the animals that received anti-NogoA treatment. The animals receiving anti-NogoA treatment showed significantly better results in terms of functional recovery, fiber tract integrity, axonal sprouting and white matter repair markers compared with the control group at 28 days. White matter integrity was in part restored by antibody-mediated inhibition of NogoA administration in those animals that were subjected to an axonal injury by subcortical stroke. This white matter restoration triggered functional recovery.

at 7 to 28 d after stroke. However, we did not observe significant differences between groups either at 7 or at 28 d after stroke; (p > 0.05) (Fig. 3C).
Axonal sprouting. Stroke was produced in the subcortical white matter of the striatum. This region contains fascicles of axons projecting from the overlying cortex. Motor cortical projections play an important role in motor function and movement. The anatomical anterograde neuronal tracer biotin dextran amine (BDA) was injected into the forelimb motor cortex 21 d after stroke, and the striatal axonal density was studied 7 d after the injection (28 d after stroke) to analyze changes in axonal sprouting after the treatment. A statistically significantly high mean intensity of BDA-labeled axons were observed in the striatum in the treated group compared with the control animals (5,497,276.06 ua ± 1,311,049.01 ua vs. 2,727,665.32 ua ± 839,278.50 ua) (p < 0.05). These results might indicate that anti-NogoA antibody treatment produces a significant axonal sprouting response from the ipsilateral cortex to the injured striatum (Fig. 3E).

Myelin restoration.
A morphological study using a HitoCryoMyelin staining kit identified the lesion zone in all the animals as a subcortical infarct affecting the white matter, with more myelinated axons in the treated animals (199.19 ± 8.17) compared with the control group (179.56 ± 7.31) (p < 0.05) (Fig. 4C).

Discussion
Blood supply disruption in stroke compromises not only some neurons but also whole axons and fibers, and therefore, brain connectivity. However, white matter injury and the mechanisms of nerve fiber (axon and myelin) repair have seldom been investigated in translational stroke research 11 . The high incidence of such damage motivates the search for an effective therapy to enhance the mechanisms underlying the repair of damaged nerve fibers after any kind of stroke. White matter repair after stroke can lead to processes that include the activation of neuronal molecular mechanisms of growth, extrinsic growth promoting factors and axon guidance signals  Rats were subjected to a subcortical stroke by endothelin I injection. Twenty-four hours later, the rats received treatment (anti-IgG or anti-NogoA antibodies). At 48 h, histological studies to determine the effectiveness of the antibody were performed. (C) Immunofluorescence showing the presence of NogoA protein in the peripheral organs (lung, liver and spleen) and the brain in both the control and the treated groups 24 h after treatment. (D) Colocalization of the NogoA protein was shown with GFAP, Olig-2, MAP-2 and IBA-1 in brain samples. (E) Quantification of immunofluorescence showed a decrease in NogoA protein in the animals that received anti-NogoA antibody. (F) Quantification of the colocalization of the NogoA protein with GFAP, Olig-2, MAP-2 and IBA-1. Abbreviations: GFAP, glial fibrillary acidic protein; Olig-2, oligodendrocyte transcription factor-2; MAP-2 microtubule-associated protein 2; IBA-1, ionized calcium-binding adapter molecule 1. (G) Biodistribution of Anti-NogoA antibody showed the antibodies (green) in the lung, liver, kidney and brain by immunofluorescence at 24 hours after i.v. administration.  in the tissue. Almost nothing is known about the optimal details of the various processes; we only know that pharmacological treatments can modify these neuronal processes. Finding a therapy in accordance with these processes (in terms of time window and doses) would be the key for a correct recovery after stroke. In this sense, myelin-associated inhibitors such us NogoA are proteins that inhibit neurite growth in adults and are thought to limit axon growth and play an inhibitory role in axonal regeneration, repair and collateral sprouting in vivo after CNS injury 4-6, 12, 13 . Thus, antibodies that target NogoA might promote axonal growth. In this study, we observed that NogoA neutralization enhanced axonal repair, tract connectivity, oligodendrogenesis and myelin formation in the striatum as well as motor functional recovery after white matter stroke in an animal model. These findings were correlated with improved motor skill recovery upon blockade of NogoA function.
Many studies have used anti-NogoA therapy as a treatment after various neurological disorders such as stroke. Most previous studies have administered NogoA antibody by intracerebral 8,14,15 or intrathecal routes [14][15][16][17][18] , using osmotic minipumps. However, to be more appropriate for translation of this treatment to clinical applications, we chose the intravenous route, which is a less invasive route previously described by another study on spinal cord injury as safe and effective 19 .
The delivery of anti-NogoA antibodies could allow their union with the protein and neutralize their negative effect on neurite and axonal outgrowth after damage. In this sense, it is important to identify in which organ (brain or other peripheral organs) the antibody anti-NogoA is acting. NogoA is enriched on the surface of oligodendrocytes in the outermost and innermost axonal myelin membrane. It is important to note that new findings showed that NogoA is not only expressed on oligodendrocytes but also in various brain areas and in several types of neurons and glial cells 20 . In our study, we observed a lower expression of NogoA protein in the brain and peripheral organs (lung, liver and spleen) compared with control animals. We also found a lower expression of NogoA protein in cells expressing Olig-2, MAP-2 and IBA-1, but not in cells expressing GFAP. These findings suggest that the anti-NogoA antibody affects not only oligodendrocytes, but also neurons, microglia and astrocytes. Moreover, the NogoA protein was also analyzed at 28 d after treatment, and the levels of this protein were lower in the treated animals compared with the controls. These results indicate that anti-NogoA injection decreases NogoA levels at least 28 d after treatment. These results are consistent with those found in a biodistribution analysis, in which anti-Nogo-A antibodies were found in the brain and in the peripheral organs (lung, liver and kidney), where the NogoA protein expression was decreased.
In a translational study, it is important to analyze whether anti-NogoA injections act on the motor dysfunctions characteristic of subcortical stroke. Previous authors found the rotarod test 21 and the mNSS test 22 to be effective in assessing the motor deficit associated primarily with subcortical stroke. In this study, anti-NogoA antibody treatment induced a significant improvement in functional recovery that was particularly notable at 28 d after treatment when compared with the controls. Although there are no previous studies that have administered anti-NogoA in a subcortical stroke model, our results are consistent with previous data showing that anti-NogoA immunotherapy improved functional recovery in an experimental animal model of middle cerebral artery occlusion 8,9,16,18,23 , and even in post-acute lesions 14 .
Some experimental studies show that a change in the reduction of lesion volume is not important to determining the severity of a cerebral infarct. In fact, infarct volume did not correlate with functional recovery when the neurovascular unit was integrated 24 . In our study, no reduction in the lesion size was observed on T2 images in animals treated with anti-NogoA immunotherapy compared with the control group at 24 h or 28 d. However, these results are consistent with previous studies that showed that this treatment did not reduce lesion size in an experimental animal model of cortical stroke 9,17,18,25 .
Stroke triggers axonal sprouting in the cortical areas adjacent to the infarct. This sprouting can be detected with anatomical mapping of cortical circuits as early as 3 weeks after stroke 26 . Axonal sprouting in the adjacent cortex after stroke occurs in the tissue immediately bordering the infarct and in motor, somatosensory and premotor areas distant from the infarct 27 . To map the fascicles of axons projecting from the overlying motor cortex that were growing after anti-NogoA treatment, neurons were anterogradely labeled using a BDA injection into the cortex 3 weeks after the stroke. Anterograde tracing with BDA demonstrated descending projections from the intact cortex. A statistically significantly high number of BDA-labeled axons were shown in the striatum of the animals that received treatment with anti-NogoA antibody compared with the control animals. These results might indicate that blocking NogoA produces a significant axonal sprouting response from the ipsilateral cortex to the injured striatum. These results correlate with those studies examining NF, given we observed that the levels of this marker were also higher in the treated animals compared with the control animals at 28 d after stroke. This result agrees with a previous study using retrograde tracing that showed a 2-to 3-fold increase in labeled neurons projecting to both sides of the spinal cord in the anti-NogoA-treated animals 28 . These results are consistent with previous data showing that the anti-NogoA antibody promotes axonal growth after various types of CNS injury 8,9,29 . Moreover, three various anti-NogoA antibodies have proven efficient in enhancing axonal regeneration and outgrowth both in vitro and in vivo 30 . Another study revealed overexpression of neuronal GAP-43, an intrinsic determinant of neuronal plasticity and a molecular indicator of axonal plasticity in the CNS 31 . Moreover, consistent with these results, other authors have demonstrated that a blockade of NogoA leads to greater synaptic plasticity and modification 32 . These results suggest that functional recovery at 28 d could be related to the process by which restructured axons, which had previously been injured, recover axon integrity and tract connectivity.
In particular, myelin-associated inhibitors are proteins thought to limit axon growth and collateral sprouting 2-6, 12, 13, 33 . Thus, antibodies that target NogoA might promote tract integrity. To analyze the fiber tract integrity after antibody anti-NogoA, we performed in vivo DTI tractography, which showed that tract thickness was recovered at 28 d after treatment compared with controls. These results suggest that blocking NogoA could promote not only axonal growth but also track integrity and connectivity. This treatment could be involved in the process by which restructured axons, which had previously been compromised and demyelinated, recover not only their proper structure but also tract connectivity. NogoA is predominantly expressed by oligodendrocytes 13 . After white matter stroke, damage to oligodendrocytes leads to white matter dysfunction through the loss of myelin. Then, enhanced proliferation, migration and differentiation of oligodendrocyte progenitor cells (OPCs) are observed in damaged and recovering white matter regions 34 . To assess whether NogoA blockage enhances oligodendrogenesis, we measured CNPase (a marker related to immature oligodendrocytes) by immunofluorescence and western blot. In our study, the levels of CNPase marker were higher in the treated animals at 28 d than in the control group.
Not only axons and oligodendrogenesis are important in the process of white matter repair. In addition, the myelin sheath allows the transmission of nerve impulses over relatively long distances, which is a process necessary for whole brain function 35 . A cryomyelin study showed an increase in myelin in those animals that received treatment with anti-NogoA compared with the control group.
In vitro experiments help to strengthen results found in an in vivo model. In agreement with previously published studies 10, 36, 37 , PC12 cells have differentiated neurites in the presence of nerve growth factor (NGF). When comparing the response of the anti-NogoA antibody group to the NGF-treated cells, the treated cells appeared to have a higher percentage of immature and mature neurites. The enhancement of neurite maturation appeared to be directly correlated with the presence of anti-NogoA at a concentration of 12 µg/ml. Our results demonstrated that although the NGF-treated cells exhibited immature neurites, the anti-NogoA antibody cells exhibited many more mature neurites. These results agree with other studies in which the pretreatment of PC12 cells with the anti-NogoA antibody was effective in blocking the inhibitory effects of the NogoA peptide in terms of neurite length, as observed for the NGF-treated group 10 . Moreover, we analyzed whether the anti-NogoA antibody was able to decrease NogoA protein levels in vitro. The results of this study showed a lower expression of the NogoA protein in PC12 cells treated with the anti-NogoA antibody compared with the control culture. Moreover, our results showed that the anti-NogoA antibody triggered an increase in neural marker expression (GAP-43, NF, MAP-2 and MBP) on PC12 cultures.
In summary, the enhancement of behavioral recovery in a variety of sensory-motor tasks, as well as anatomical evidence of fiber growth, tract connectivity and oligodendrogenesis, have been reported after anti-NogoA antibody administration in a subcortical stroke animal model.

Materials and Methods
In vitro studies. Effectiveness of anti-NogoA blocking NogoA. To study whether the anti-NogoA antibody induced a block of NogoA expression, an in vitro study using PC12 cells was performed. PC12 cells (Sigma-Aldrich, catalogue number 88022401) were maintained in Dulbecco's Modified Eagle's Medium (DMEM; Invitrogen) high glucose, supplemented with 10% horse serum (Sigma-Aldrich), 10% fetal bovine serum (Biowest) and 1% penicillin/streptomycin (P/S) at 5% CO2, at 37 °C. For the NGF treatment (NGF-7S, Sigma-Aldrich) to induce neurite and axonal outgrowth in PC12 cells, they were plated onto poly-l-lysine (Sigma-Aldrich) coated dishes and maintained in DMEM containing 1% horse serum, 1% P/S and 100 ng/mL NGF. After 14 d, anti-NogoA antibody (12 µg/mL, Immunostep S.L.) was incubated with half the cultures for 72 h (Fig. 1A). Next, the expression of NogoA protein was determined by immunofluorescence using the NogoA antibody (1:100, Abcam). A secondary antibody for immunofluorescence was goat anti-mouse Alexa Fluor 488. The slices were examined using a LEICA TCS SPE spectral confocal microscope (Leica Microsystems, Heidelberg, Germany), and the confocal images were analyzed using LEICA software LAS AF, version 2.0.1, Build 2043.
Axonal sprouting. After 72 h of incubation with anti-NogoA antibody, changes in morphology and neurite outgrowth of PC12 cells were studied using a Nikon Eclipse-Ti inverted microscope and NIS-elements software. In vivo studies. Ethics statement. All the procedures were performed in accordance with stroke therapy academic industry roundtable 38,39 and RIGOR 40 relevant guideline recommendations in terms of randomization, blinding and statistical powering. All the experimental protocols were approved by the medical school's Ethics Committee for the Care and Use of Animals in Research of La Paz University Hospital, according to Spanish and European Union rules (86/609/CEE and RD53/2013). This committee includes endpoint criteria. All the animals received a postsurgery injection of meloxicam as analgesia, and we have a protocol for the daily supervision of animals. If any animals show pain symptoms, they again receive meloxicam (2 mg/kg) as analgesia.
Animals and surgical procedure. To provoke white matter injury, a subcortical ischemic stroke was induced in male Sprague-Dawley rats (200-250 g, Charles River Laboratories). Anesthesia was induced using an anesthetic chamber with 5% isoflurane in a 5-L/min oxygen flow, and the animals were maintained using a face mask with 3% isoflurane in a 2-L/min oxygen flow. In all the animals, the femoral artery was cannulated during surgery and induction of cerebral ischemia. The cannulation allowed for continuous monitoring of physiological parameters including blood glucose levels, blood gases and blood pressure (Omicron ALTEA Monitor; RGB Medical Devices, Madrid, Spain). After an intraperitoneal injection of meloxicam (2 mg/kg), the animals were placed in a stereotactic frame. A craniotomy was performed adjacent to the bregma and intrastriatal white matter injury was induced by injection of 1 µL of endothelin-1 (ET-1; Calbiochem) (0.25 µg/µL) for subcortical ischemic stroke.
Anti-NogoA formation. On day 0, NogoA protein and the appropriate adjuvants were injected into the host, which were recognized by the immune system as foreign and were targeted by antibodies for immune blockage. All antigen-presenting cells internalize antigens by endocytosis or phagocytosis. Recognition of antigens induces the release of stimulatory signaling, thus activating B cells. Activated B cells divide to create both memory B-cell and plasma cell populations. Plasma cells secrete antibodies into the serum for immune recognition, whereas memory B-cells persist for longer, thus providing a quick response to secondary exposure from the same antigen. This process is termed immunization. Fourteen days after the first immunization, the second immunization was performed, again injecting the NogoA protein and the appropriate adjuvants. At 24 d, serum from the rabbit was collected. At 35 d after first immunization, a third immunization was performed, and serum was collected at 45 d. Finally, a fourth immunization was performed at 56 d, and serum was collected at 66 d. Measuring the antibody levels by ELISA during the immunization process is required to evaluate progress. Because many antigen-specific antibodies are secreted by multiple activated B-cells, anti-NogoA antibodies need to be isolated from the other antibodies ( Fig. 2A).
Additionally, 8 animals were used in order to test the effectiveness of the anti-NogoA injection to block rat NogoA protein. All the animals were subjected to stroke, and 24 h later, 4 animals received anti-NogoA and 4 animals received anti-IgG. These animals were euthanized 24 h after treatment. The brains and peripheral organs (liver, lung, kidney and spleen) were processed to perform immunofluorescence to analyze the levels of the NogoA protein in the various tissues (Fig. 2B).
Treatment was administered via the tail vein 24 h after surgery, and the remaining animals were euthanized at 28 d (Fig. 3A). Five rats were excluded from the study because 2 rats died during surgery and 3 rats died during the magnetic resonance procedure (2 from the control group and 1 from the treated group).

Effectiveness of anti-NogoA in terms of protein neutralization.
To study whether the anti-NogoA antibody induced a block of NogoA protein in vivo, the antibody was intravenously administered 24 h after the animals suffered a stroke. The expression of NogoA protein was determined by immunofluorescence using NogoA antibody (1:100, Abcam) in the brain and in the peripheral organs (lung, liver, kidney and spleen) 24 h after anti-NogoA administration. To determine in which type of cells the expression of NogoA was blocked, colabeling was performed with the NogoA antibody (1:100, Abcam) and GFAP (1:400, Chemicon), MAP-2 (1:1000, Milipore), Olig-2 (1:500, Millipore) and IBA-1 (1:1000, Milipore) in the brain samples. Secondary antibodies for immunofluorescence were goat anti-mouse Alexa Fluor 488 and anti-rabbit Alexa Fluor 488/594 (1:750, Invitrogen). The slices were examined using a LEICA TCS SPE spectral confocal microscope (Leica Microsystems), and the confocal images were analyzed using LEICA software LAS AF, version 2.0.1, Build 2043. Moreover, the biodistribution of the anti-NogoA antibody was studied after incubating the slides of the brain and peripheral organs in secondary antibody anti-rabbit Alexa Fluor 488 (1:750, Invitrogen).
Functional evaluation scales. Functional evaluations were performed on all the animals by a blinded observer before surgery and after 48 h, 7 d, 14 d and 28 d. Motor performance was evaluated using the rotarod test and the mNSS. The rotarod test measured the time to falling from a rotating cylinder 21 . The mNSS test was used to measure sensory and motor deficits; it is a composite of motor, sensory, balance and reflex tests, and neurological function is graded on a scale of 0 to 18. A score of 0 is associated with normal neurological function, and a score of 18 represents the maximum functional deficit. For the severity scores of injury, 1 point is awarded for a specific abnormal behavior or for the lack of a tested reflex; thus, the more injured the animals, the higher the score on the test [41][42][43] . The researchers recording the data were entirely blinded to the experimental groups.
In vivo magnetic resonance imaging. Lesion sizes were analyzed after 7 d and 28 d by magnetic resonance imaging (MRI) using a 7-Tesla horizontal bore magnet (Bruker PharmaScan, Ettlingen, Germany) and T2-weighted images. The lesion area was expressed as a percentage of the contralateral hemisphere. For the tractography analysis, a diffusion-weighted MRI was acquired on a Bruker PharmaScan system (Bruker Medical Gmbh, Ettlingen, Germany) using a 7.0-T horizontal-bore superconducting magnet, equipped with a 1 H circular polarized volume coil with inner diameter of 40 mm and a Bruker gradient insert of 90-mm diameter (maximum intensity 30 G/ cm). The animals were anesthetized with a 2% isoflurane-oxygen mixture in an induction chamber, and the flow of anesthetic gas was continuously regulated to maintain a breathing rate of 50 + /− 20 bpm. The animals' temperature was maintained at approximately 37 °C with a circulating warm water blanket. The physiological state of the rats was monitored using a monitoring system by SA instruments (Stony Brook, NY) that controlled the respiratory rate and body temperature. Diffusion tensor data were acquired with a spin echo single shot echo planar imaging (EPI) pulse sequence using the following parameters: TR/TE 8000/35 ms; a signal average of 12, 30 noncollinear diffusion gradient scheme with a diffusion weighting b = 500 s/mm 2 and b = 1000 s/mm 2 , 18 slices with a slice thickness of 1.5 mm without a gap, field of view 35 × 35 mm. Total imaging time was 1 h 44 min. All the EPI data were acquired with a single shot EPI sequence, 96 × 96 matrix, and zero filled-in k space to construct a 128 × 128 image matrix. Fractional anisotropy, mean diffusivity, trace and the eigenvalues and eigenvector maps were calculated with a homemade software application written in Matlab (R2007a); the 3D fiber tract map was created using MedINRIA DTI Track software, as previously described 41,43 . The researchers recording the data were entirely blinded to the experimental groups.
Mapping of motor cortex connections. Changes in axonal sprouting after treatment were assessed using an intracortical injection of the anterograde neuronal tracer BDA (10000 MW, Life Technologies, Grand Island, NY, USA). The stereotaxic coordinates of the injection site with respect to the bregma were as follows: 0.04 mm posterior, 0.35 mm lateral and 0.16 mm ventral. The rat cortexes were injected with BDA at 21 d after stroke and they were euthanized 28 d after stroke. All the brains were fixed, frozen and coronally sectioned at 40 µm. The sections were visualized using a confocal microscope (Leica Microsystems). Mean intensity of striatal cells labeled with BDA was quantified using 4 animals, with 10 sections from each animal per group. The researchers recording the data were entirely blinded to the experimental groups.
Immunohistochemistry, immunofluorescence and western blot analyses. Frozen sections were stained using the CryoMyelin Kit (Hitobiotech), which allows sensitive localization and visualization of myelin fibers. The mean intensity of myelin staining in the region of interest was quantified using a Nikon Eclipse-Ti inverted microscope and NIS-elements software. The lesion area was studied in detail using immunofluorescence and western blot analyses. The various white matter-associated antibodies used for immunofluorescence and western blot analyses were CNPase (1:500, Sigma-Aldrich), NF (1:100, Dako) and NogoA (1:100, Abcam), followed by goat anti-mouse and anti-rabbit Alexa Fluor 488 (1:750, Invitrogen). For the western blot analysis, the units were normalized based on Β-actin (1:400, Sigma-Aldrich). To quantify the expression of white matter-associated markers, the mean fluorescence intensity was evaluated using a 40X objective lens (4 animals in each group, 5 sections in each animal per group, 10 different microscope fields). The experiments, images and quantification of the samples were performed by blinded observers, using the same microscope configurations, to eliminate bias due to background normalization.
Statistical analysis. Results were expressed as mean ± standard deviation. The data were compared using the Kruskal-Wallis test followed by the Mann-Whitney test. Values of p < 0.05 were considered significant at a 95% confidence interval; the data were calculated using statistical SPSS 16 and GraphPad software. The power analysis showed that with nonparametric testing, at least 10 animals needed to be randomized to each group for a significance level of 5% (alpha) and a potency of 80% (1-beta) for functional evaluation.