RGMa inhibition with human monoclonal antibodies promotes regeneration, plasticity and repair, and attenuates neuropathic pain after spinal cord injury methods

[
  {
    "@context": "https://schema.org",
    "@type": "HowTo",
    "name": "RGMa inhibition with human monoclonal antibodies promotes regeneration, plasticity and repair, and attenuates neuropathic pain after spinal cord injury methods",
    "description": "Evidence-backed execution summary for RGMa inhibition with human monoclonal antibodies promotes regeneration, plasticity and repair, and attenuates neuropathic pain after spinal cord injury methods from RGMa inhibition with human monoclonal antibodies promotes regeneration, plasticity and repair, and attenuates neuropathic pain after spinal cord injury.",
    "totalTime": "PT77280M",
    "step": [
      {
        "@type": "HowToStep",
        "position": 1,
        "name": "Human anti-RGMa mabs promote neurite outgrowth in vitro",
        "text": "Previous experiments showing that RGMa neutralization promotes functional recovery after hemisection SCI were performed using rabbit polyclonal antibodies. Hence, the development of a clinically relevant monoclonal antibody for human RGMa that promotes regeneration has become the target of intense investigation. The human antibodies AE12-1 and AE12-1Y are high-affinity RGMa-selective mabs with comparable binding to human, rat and mouse RGMa. The 2 mabs differ in their half-life, with AE12-1 having a longer half-life of 6 days compared to AE12-1Y half-life of 2 days. As shown previously, the monoclonal antibodies are specific for RGMa. In Western blots of mouse cortical neuron lysates, both mabs are specific for RGMa and no cross-reactivity was observed between other RGM members (Fig. ). Cultured mouse primary cortical neurons also expressed RGMa, as shown by immunostaining with..."
      },
      {
        "@type": "HowToStep",
        "position": 2,
        "name": "RGMa human antibodies are detected in rat serum, CSF, and injured spinal cord",
        "text": "Factors limiting potential treatments for SCI include mode of administration of the therapeutic. As intrathecal administration via osmotic pumps and catheters may result in secondary tissue damage or associated problems such as catheter occlusion, we performed systemic administration of the RGMa mabs. A summary of the study design is depicted in Fig.. Rats were pre-trained and then clip impact-compression SCI was made at T8, immediately followed by local intraspinal and intravenous (i.v.) injections of the mabs or controls (hIgG isotype control or PBS (phosphate buffered saline) vehicle). The i.v. injections were repeated weekly for 6 weeks. At 6 weeks post-SCI, cerebrospinal fluid (CSF) was sampled via lumbar puncture and the tracer biotinylated dextan amine (BDA) was injected into the sensorimotor cortex for anterograde labeling of the corticospinal tract (CST). At 9 weeks po..."
      },
      {
        "@type": "HowToStep",
        "position": 3,
        "name": "RGMa mabs improve functional recovery after SCI",
        "text": "We then examined the effect of inhibiting RGMa with AE12-1 and AE12-1Y after acute impact-compression SCI. Neurological recovery was monitored weekly using the BBB locomotor rating scale, motor subscore, and horizontal ladderwalk test. Acute treatment with AE12-1 showed significant recovery of the BBB as early as 1 week post-SCI compared to PBS or hIgG controls which was maintained for the duration of the study (Fig. ). In contrast, AE12-1Y showed a delayed improvement on the BBB with a statistically significant difference at 6 weeks post-SCI relative to controls (12.6 vs 9.9 PBS). The difference in recovery profiles of AE12-1 and AE12-1Y may be due to the longer half-life of AE12-1. Both mabs also showed a trend towards a higher motor subscore compared to controls (Fig. ). We also assessed hindlimb coordination with the ladderwalk test in which footfall errors are scored,..."
      },
      {
        "@type": "HowToStep",
        "position": 4,
        "name": "RGMa mabs enhance neuronal survival",
        "text": "In a rat model of optic nerve injury, inhibiting the function of the Neogenin receptor promoted neuronal survival. Previously we showed that inhibiting Neogenin function with a peptide approach enhanced perilesional neuronal sparing. Membrane fractionation experiments also demonstrated that injection of AE12-1Y altered the localization of Neogenin to the heavy membrane fraction, thus preventing the association of Neogenin with lipid rafts and blocking cell death signaling. Therefore, we examined if neutralizing RGMa with the monoclonal antibodies could attenuate neuronal loss after SCI. After 6 weeks of AE12-1 or AE12-1Y treatment, perilesional neurons were quantified with the neuronal marker NeuN at 9 weeks post-SCI. Treatment with the RGMa antibodies increased the number of perilesional neurons approximately 1.5-fold compared to controls (Fig. ). To determine if the neurona..."
      },
      {
        "@type": "HowToStep",
        "position": 5,
        "name": "Treatment with anti-RGMa mabs promote axonal regeneration",
        "text": "We examined the effect of inhibiting RGMa with the mabs on axonal regeneration. We quantified the mean number of 5HT+ serotonergic fibers caudal to the lesion site, and found a significantly higher number of 5HT+ fibers in AE12-1 treated rats relative to controls (Fig. ). Rats injected with AE12-1Y showed a trend towards a higher number of 5HT labeled axons although there was significant variability in the number of 5HT fiber counts in the AE12-1Y groups as reflected in the large standard error. Furthermore, we also examined the effect of RGMa neutralization on descending CST pathways. We injected BDA tracer into the sensorimotor cortex to anterogradely label the CST. Our SCI model of impact-compression injury in which both the dorsal and ventral aspects of the spinal cord are simultaneously compressed results in central cavitation of the gray matter and adjacent whi..."
      },
      {
        "@type": "HowToStep",
        "position": 6,
        "name": "RGMa mabs attenuate neuropathic pain",
        "text": "We assessed the effect of RGMa neutralization on the development of neuropathic pain following SCI. Interestingly, we found that mab treatment reduced at-level mechanical allodynia and thermal hyperalgesia. At 6 weeks post-SCI, rats administered AE12-1 showed significantly fewer adverse responses to the 4 g vonFrey stimulus compared to controls (Fig. ). Rats treated with either AE12-1 or AE12-1Y showed reduced latency to withdrawal of the tail in response to a heat stimulus compared to controls (Fig. ). We then examined the correlation of Iba-1 expression in the spinal cord, but found no significant difference in the percentage area of Iba-1 + staining rostral or caudal immediately adjacent to the lesion site (data not shown). This quantitation included all Iba-1 immunostained cells, including both activated microglia and macrophages. However, Iba-1..."
      },
      {
        "@type": "HowToStep",
        "position": 7,
        "name": "Western blots",
        "text": "Mouse cortical neurons were lysed in RIPA buffer containing a protease inhibitor cocktail. Samples were denatured at 95 °C for 5 min in a 6X Lamelli buffer (50 mM Tris-HCl [pH6.8], 2% SDS, 10% glycerol, 1% β-mercaptoethanol, 12.5 mM EDTA, and 0.02% bromophenol blue). Samples were loaded on a 10% acrylamide gel in Tris-Glycine running buffer (25 mM Tris, 192 mM Glycine, and 0.1% SDS) and transferred onto a nitro-cellulose membrane. Blots were blocked with 5% nonfat dry milk in PBS, and probed with the anti-RGMa mabs (AE12-1; 1:1000 (1 mg/ml) and AE12-1Y; 1:1000 (1 mg/ml)) and anti-Neogenin (E20; Santa Cruz; 10 mg/ml) at 4 °C overnight. After three washes in PBS containing 0.1% Tween-20, blots were probed with an Odyssey goat anti-mouse secondary antibody (1:4000; Li-COR, Lincoln, Ne, USA) for 2hr at room tempera..."
      },
      {
        "@type": "HowToStep",
        "position": 8,
        "name": "Spinal cord injury and injections",
        "text": "Rats were anesthetized by inhalation of 2% isofluorane in combination with a mixture of nitrous oxide and oxygen (1:2, v/v). Under aseptic conditions, a laminectomy was performed at level T9/10. A clip impact-compression injury was made at spinal cord level T8 with a 20 g force for 1 min with a modified aneurysm clip as reported previously,. Briefly, and as illustrated in Fig., the clip is applied extradurally producing a bilateral impact force followed by sustained dorsal-ventral compression, a clinically relevant model of SCI reflecting human pathology,. Immediately following SCI, rats were intraspinally injected with either AE12-1, AE12-1Y, hIgG isotype control, or PBS vehicle. There were four groups of rats: 1) AE12-1 (n = 10), AE12-1Y (n = 11), hIgG (n = 11), and PBS (n = 8). The experimental protocol is summa..."
      }
    ],
    "tool": [
      {
        "@type": "HowToTool",
        "name": "RGMa human antibodies are detected in rat serum, CSF, and injured spinal cord"
      },
      {
        "@type": "HowToTool",
        "name": "RGMa mabs improve functional recovery after SCI"
      },
      {
        "@type": "HowToTool",
        "name": "Generation of human RGMa monoclonal antibodies"
      },
      {
        "@type": "HowToTool",
        "name": "Assay for measuring antibody levels in serum and CSF"
      },
      {
        "@type": "HowToTool",
        "name": "Spinal cord injury and injections"
      },
      {
        "@type": "HowToTool",
        "name": "BBB and motor subscore"
      },
      {
        "@type": "HowToTool",
        "name": "Ladderwalk"
      },
      {
        "@type": "HowToTool",
        "name": "CatWalk"
      }
    ],
    "supply": [
      {
        "@type": "HowToSupply",
        "name": "Human anti-RGMa mabs promote neurite outgrowth in vitro"
      },
      {
        "@type": "HowToSupply",
        "name": "RGMa human antibodies are detected in rat serum, CSF, and injured spinal cord"
      },
      {
        "@type": "HowToSupply",
        "name": "Generation of human RGMa monoclonal antibodies"
      },
      {
        "@type": "HowToSupply",
        "name": "Assay for measuring antibody levels in serum and CSF"
      },
      {
        "@type": "HowToSupply",
        "name": "Western blots"
      },
      {
        "@type": "HowToSupply",
        "name": "In vitro neuronal immunostaining and neurite outgrowth assay"
      },
      {
        "@type": "HowToSupply",
        "name": "Histology and immunostaining"
      },
      {
        "@type": "HowToSupply",
        "name": "Double-label NeuN/TUNEL staining"
      }
    ],
    "isBasedOn": {
      "@type": "ScholarlyArticle",
      "headline": "RGMa inhibition with human monoclonal antibodies promotes regeneration, plasticity and repair, and attenuates neuropathic pain after spinal cord injury",
      "datePublished": "2017",
      "author": [
        {
          "@type": "Person",
          "name": "Andrea J. Mothe"
        },
        {
          "@type": "Person",
          "name": "Nardos G. Tassew"
        },
        {
          "@type": "Person",
          "name": "Alirezha P. Shabanzadeh"
        },
        {
          "@type": "Person",
          "name": "Romeo Penheiro"
        },
        {
          "@type": "Person",
          "name": "Robin J. Vigouroux"
        },
        {
          "@type": "Person",
          "name": "Lili Huang"
        },
        {
          "@type": "Person",
          "name": "Christine Grinnell"
        },
        {
          "@type": "Person",
          "name": "Yi-Fang Cui"
        },
        {
          "@type": "Person",
          "name": "Emma Fung"
        },
        {
          "@type": "Person",
          "name": "Philippe P. Monnier"
        },
        {
          "@type": "Person",
          "name": "Bernhard K. Mueller"
        },
        {
          "@type": "Person",
          "name": "Charles H. Tator"
        }
      ],
      "identifier": "10.1038/s41598-017-10987-7"
    }
  },
  {
    "@context": "https://schema.org",
    "@type": "BreadcrumbList",
    "itemListElement": [
      {
        "@type": "ListItem",
        "position": 1,
        "name": "Experiments",
        "item": "https://replicatescience.com/experiments"
      },
      {
        "@type": "ListItem",
        "position": 2,
        "name": "RGMa inhibition with human monoclonal antibodies promotes regeneration, plasticity and repair, and attenuates neuropathic pain after spinal cord injury methods",
        "item": "https://replicatescience.com/experiments/rgma-inhibition-with-human-monoclonal-antibodies-promotes-regeneration-plasticity-and-repair-and-attenuates-neuropathic-pain-after-spinal-cord-injury-methods-andrea-j-mothe-pmc5585/rgma-inhibition-with-human-monoclonal-antibodies-promotes-regeneration-plasticity-and-repair-and-att-mlph973l"
      }
    ]
  }
]