Reduced inflammation accompanies diminished myelin damage and repair in the NG2 null mouse spinal cord
© Kucharova et al; licensee BioMed Central Ltd. 2011
Received: 5 October 2011
Accepted: 13 November 2011
Published: 13 November 2011
Multiple sclerosis (MS) is a demyelinating disease in which blood-derived immune cells and activated microglia damage myelin in the central nervous system. While oligodendrocyte progenitor cells (OPCs) are essential for generating oligodendrocytes for myelin repair, other cell types also participate in the damage and repair processes. The NG2 proteoglycan is expressed by OPCs, pericytes, and macrophages/microglia. In this report we investigate the effects of NG2 on these cell types during spinal cord demyelination/remyelination.
Demyelinated lesions were created by microinjecting 1% lysolecithin into the lumbar spinal cord. Following demyelination, NG2 expression patterns in wild type mice were studied via immunostaining. Immunolabeling was also used in wild type and NG2 null mice to compare the extent of myelin damage, the kinetics of myelin repair, and the respective responses of OPCs, pericytes, and macrophages/microglia. Cell proliferation was quantified by studies of BrdU incorporation, and cytokine expression levels were evaluated using qRT-PCR.
The initial volume of spinal cord demyelination in wild type mice is twice as large as in NG2 null mice. However, over the ensuing 5 weeks there is a 6-fold improvement in myelination in wild type mice, versus only a 2-fold improvement in NG2 null mice. NG2 ablation also results in reduced numbers of each of the three affected cell types. BrdU incorporation studies reveal that reduced cell proliferation is an important factor underlying NG2-dependent decreases in each of the three key cell populations. In addition, NG2 ablation reduces macrophage/microglial cell migration and shifts cytokine expression from a pro-inflammatory to anti-inflammatory phenotype.
Loss of NG2 expression leads to decreased proliferation of OPCs, pericytes, and macrophages/microglia, reducing the abundance of all three cell types in demyelinated spinal cord lesions. As a result of these NG2-dependent changes, the course of demyelination and remyelination in NG2 null mice differs from that seen in wild type mice, with both myelin damage and repair being reduced in the NG2 null mouse. These studies identify NG2 as an important factor in regulating myelin processing, suggesting that therapeutic targeting of the proteoglycan might offer a means of manipulating cell behavior in demyelinating diseases.
KeywordsInflammation myelin repair NG2 ablation oligodendrocyte progenitors pericytes macrophages
During the acute phase of multiple sclerosis (MS), damage to the blood-brain barrier allows infiltration of blood-derived cells that cause disruption of the myelin sheath [1–5]. The capability of the CNS for myelin repair is mediated by the action of oligodendrocyte progenitor cells (OPCs), which not only generate oligodendrocytes during CNS development, but also persist as the largest cycling population in the mature CNS [6–9]. These "adult" OPCs serve as a source of cells for myelin repair [8, 10–12], but also exhibit other functions of mature glia , including contributions to nodes of Ranvier [14–16] and reception of synaptic input [17, 18]. OPC function and remyelination of axons nevertheless often fail in both relapsing-remitting and progressive MS [19–21]. The inability of OPCs to produce adequate numbers of myelinating oligodendrocytes has been attributed to several factors, including failure of OPC proliferation, failure of OPC recruitment to the lesion, failure of OPC differentiation, and failure of OPCs or oligodendrocytes to interact with neurons. Compounding this complexity, MS is a multifactorial disease, involving participation of multiple factors in both myelin damage and myelin repair. A better understanding of the molecular mechanisms of myelin degradation and regeneration is clearly required for improved treatment of this primary demyelinating disease.
Here we show that the NG2 proteoglycan is expressed by three cell types that invade demyelinated CNS lesions: OPCs, macrophages/microglia, and microvascular pericytes. In addition to serving as a marker for these cell types [22, 23], NG2 also promotes cell proliferation and motility. In the neonatal NG2 null mouse, decreased OPC proliferation reduces the pool of progenitors available for generating myelinating oligodendrocytes, resulting in reduced developmental myelination in the cerebellum . Ablation of NG2 also causes deficits in pericyte function. Decreased pericyte recruitment and interaction with endothelial cells lead to diminished vascularization in both ocular and tumor models in the NG2 null mouse [25, 26]. We therefore have the ability to investigate the role of NG2 in multiple cell types during the processes of demyelination and remyelination.
Following microinjection of L-α-lysolecithin into the spinal cord white matter, we have investigated the activation, proliferation, recruitment, and maturation of cells that are normally NG2-positive in the wild type mouse. The importance of the NG2 molecule and NG2-positive cells in demyelination and remyelination has been evaluated via comparisons of wild type and NG2 knockout animals. The absence of NG2 causes significant deficits in the behavior of OPCs, macrophages/microglia, and pericytes, accompanied by quantitative changes in the phenomena associated with axon demyelination and remyelination.
Animal work was performed according to guidelines issued by the National Institutes of Health, following procedures approved by the Office of Laboratory Animal Welfare. All experimental protocols were approved by the Sanford-Burnham Institutional Animal Care and Use Committee. The current experiments utilized male wild type (NG2+/+) and NG2 null (NG2-/-) mice between the ages of 3-5 months. NG2 null mice were generated by a homologous recombination strategy and backcrossed for 10 generations onto the C57Bl/6 background .
Lysolecithin-induced demyelination in the spinal cord of mice
For spinal cord surgery, male mice (28-38 g) were anesthetized with Ketamine/Xylazine (100/10 mg/kg) administered intraperitoneally. Depth of anesthesia was assured by monitoring lack of response to a noxious foot pinch prior to commencing surgery. A skin incision was made above the lower thoracic vertebrae. Paravertebral muscles on both sides of the Th11-L1 vertebrae were cut, and the vertebral column was stabilized with transverse process clamps (Stoelting). The spinal cord was exposed between the Th12-Th13 vertebrae, and a small incision was made in the dura just lateral to the posterior spinal vein. A 1.5 μl solution of 1% L-α-lysolecithin (Lysophosphatidylcholine; Sigma, St. Louis, MO) in 0.1 M phosphate buffer was injected 0.5 mm deep into the dorsal column at a rate of 0.75 μl/minute. This was accomplished using a micromanipulator (Stoelting, Wood Dale, IL), 32 G needle, 5 μl syringe (7762-05, 87930; Hamilton), and digital injector (Harvard Apparatus, Holliston, MA). As a sham control, injections were done with 0.1 M PBS. The needle was left in place for an additional 2 min to avoid backflow of the lysolecithin or PBS. The muscle and skin incisions were sutured with gut and nylon, respectively (Harvard apparatus). In order to reduce postoperative pain after recovery from anesthesia, animals received a subcutaneous injection of buprenorphine (1.0 mg/kg).
Tissue preparation and immunocytochemistry
Some animals received intraperitoneal doses of 5-bromo-2-deoxyuridine (BrdU, 80 mg/kg) on post-surgery day 4, three days prior to euthanasia at day 7. At 1, 2, and 6 weeks after lysolecithin injection, animals were deeply anesthetized with Ketamine/Xylazine (100/10 mg/kg) and transcardially perfused with 0.1 M PBS, followed by 4% paraformaldehyde (pH 7.4). Spinal cords were removed and post-fixed for 24 hours at 4°C in the same fixative used for transcardial perfusion. Spinal cords were cryoprotected for 24 hours at 4°C in 0.1 M phosphate buffer containing 20% sucrose. Transverse sections (30 μm) were cut at -16°C on a cryostat microtome (Cryocut, 1800), and collected free-floating in 0.1 M PBS containing 0.02% sodium azide.
For immunostaining, free-floating sections were first incubated for 60 min at room temperature in 0.1 M PBS containing 5% normal goat serum and 0.5% Triton X-100. Sections were then incubated overnight at 4°C with primary antibodies diluted in PBS containing 0.8% Triton X-100, 0.02% sodium azide, and 5% normal goat serum. The following primary antibodies were used: 1) guinea pig anti-NG2 (1:25; ); 2) rabbit anti-PDGFRα (1:100; ); 3) rat anti-BrdU (OBT0030G, Serotec, 1:50); 4) mouse anti-Pan-Axonal Neurofilament (smi-312R, Sternberger, 1:500); 5) mouse or rabbit anti-myelin basic protein (MBP, Sternberger MSMI 94, 1:500 or Chemicon, AB980 1:100); 6) rabbit anti-PDGFRβ (1:100; ); 7) rat anti-mouse CD11b (550282, BD Pharmingen); 8) rabbit anti-IBA-1 (019-19741, Wako). After three 10-min washes with PBS, the sections were incubated with appropriate combinations of secondary antibodies: goat anti-mouse (Alexa 488; A11029, Invitrogen), anti-rabbit (Alexa 568; A11036 or Alexa 647; A21245, Invitrogen), donkey anti-guinea pig (Cy2 or Cy3; 706-225-148 or 706-165-148, Jackson ImmunoResearch), and/or goat anti-rat (Alexa 488; A11006, Invitrogen). Secondary antibodies were diluted 1:250 in the same solution as the primary antisera. In the case of BrdU, sections were incubated in 2N HCl for 30 min at 37°C, followed by boric acid neutralization (pH 8.5) for 10 min, and then processed via the immunostaining protocol described above. 4'-6-diamidino-2-phenylindole (DAPI, 4 μg/mL, D3571, Invitrogen) was used for general nuclear staining of all sections. After washing three times for 10 min with PBS, the sections were mounted on slides, air-dried, and then cover-slipped with Vectashield (H-1000, Vector lab).
In some cases myelin was also visualized histochemically in 5 μm thick paraffin sections using Kiernan's Eriochrome Cyanin technique , coupled with counterstaining by Nuclear Fast Red (H-3403, Vector lab).
Quantitative RT-PCR analysis
Primer sequences used in qRT-PCR
5'-CCA GTA TGA CTC CAC TCA CG-3'
5'-GAC TCC ACG ACA TAC TCA GC-3'
5'-TGC TGA TGG GAG GAG ATG TCT-3';
5'-TTT CTT TCA GGG ACA GCC TGT T-3';
5'-AGG TCA CAG GAG AAG GGA CGC C-3'
5'-TGC GAA GCA CCT TGG AAG CCC-3'
5'-CTG GAC AAC ATA CTG CTA ACC G-3'
5'-GGG CAT CAC TTC TAC CAG GTA A-3'
5'-GCC CAT CCT CTG TGA CTC AT-3'
5'-AGG CCA CAG GTA TTT TGT CG-3'
Image processing and quantification
At least 4 wild type and 4 NG2 null male mice were examined at each time point for quantitative analyses of various aspects of demyelination and remyelination. For calculation of demyelination volume, every 10th section from a 6 mm segment of spinal cord (i.e., a total of twenty 30 μm sections spanning from 3 mm above to 3 mm below the injection site) was immunostained for MBP. A Nikon fluorescence microscope was used to acquire images of each section, allowing determination of individual areas of demyelination (mm2) via image analysis (Image Pro Plus 5.1; Media Cybernetics). Each individual value was multiplied by 10 to obtain the demyelinated volume for that particular segment of 10 sections, and all 20 values were then summed to obtain the total volume of demyelination. For animals of the same genotype and survival period, an average volume of demyelination was obtained and expressed as a mean value ± SD.
The location and abundance of PDGFRα, PDGFRβ, and CD11b immunoreactive cells in the dorsal column were analyzed in 7 sections spanning 1 mm of the central part of the demyelinated lesion. Immunostained sections were scanned via confocal microscopy (FV 1000 and FV10-ASW Ver. 2.0, Olympus). From each scan, we assembled a z-stack of 11 optical sections, each separated by 1 μm. Data from each of the z-stacks were averaged to yield values for the density of immunoreactive cells.
Colocalization of PDGFRα, PDGFRβ, CD11b, or IBA-1 immunoreactivity with immunostaining for either NG2 or BrdU was analyzed in a single optical section obtained from each of 7 sections. For these double labeling studies, the threshold for image capture was set high enough to avoid low levels of diffuse staining due to the presence of proteolytically shed NG2. This allowed us to focus on localization of cell surface NG2. Mitotic indices for PDGFRα, PDGFRβ and IBA-1 immunoreactive cells were calculated as the percentage of BrdU-positive cells in each of the three cellular populations.
Throughout the various analyses, images were processed with Adobe Photoshop CS3 Ver. 10.0 (Adobe Systems) to standardize brightness and contrast. All data were analyzed statistically using ANOVA and un-paired t-tests. P-values less than 0.05 were considered statistically significant.
NG2 expression in wild type animals following lysolecithin injection
Abundance of NG2, PDGFR alpha, CD11b, and PDGFR beta expressing cells in wild type and NG2 null mice 1, 2, and 6 weeks after lysolecithin injection.
6.12 ± 1.7c
8.32 ± 2.1c
6.62 ± 0.9c
6.77 ± 2.6c
4.82 ± 1.4c
100 ± 8.2
100 ± 11.5
100 ± 9.1
100 ± 17.7
72.69 ± 16.8*
33.23 ± 10.3***
60.59 ± 15*
75.45 ± 6b
111.4 ± 1.9
174.83 ± 18.6c
121.71 ± 18.7
89.7 ± 6.6***
103.13 ± 16.6c**
87.71 ± 12.4a*
23.99 ± 11.7c
33.01 ± 4.9c
6.41 ± 0.8c
33.83 ± 2.6c
40.8 ± 6.4a
4.21 ± 1.3c*
44.69 ± 7*
Lysolecithin-induced demyelination in wild type and NG2 null mice
Effects of NG2 ablation on abundance of specific cell types during demyelination and remyelination
Along with comparisons of demyelination and remyelination in wild type and NG2 null mice, we evaluated the recruitment and abundance of specific cell types during the injury and repair processes. We focused on OPCs, macrophages/microglial cells, and pericytes; i.e. the cells in wild type mice that express NG2 under physiological or pathological conditions.
Six weeks after lysolecithin injection, CD11b-positive macrophages/microglial cells are still seen less frequently in NG2 null lesions than in wild type lesions (Figures 3G and 3H). However, PDGFα-positive OPCs (Figures 3C and 3D) and PDGFRβ-positive pericytes (Figures 3K and 3L) now appear to be more abundant in NG2 null lesions than in wild type lesions. We believe this is due to delayed recruitment of immature OPCs and pericytes in the absence of NG2. In wild type animals, maturing cells recruited at earlier time points may have already down-regulated expression of the PDGFRα and PDGFRβ markers.
Effect of NG2 ablation on cytokine expression
Effects of NG2 ablation on cell proliferation and motility
Proliferation of PDGFRα, PDGFRβ, and IBA-1 expressing cells in wild type and NG2 null mice.
Total Pα positive cells
178.23 ± 20.3
127.94 ± 18.4*
Pα/BrdU positive cells
26.4 ± 2.2
10.76 ± 2.9
14.81 ± 3.1%
8.41 ± 0.6%**
Total Pβ positive cells
29.8 ± 5.7
21.91 ± 3.4*
Pβ/BrdU positive cells
4.53 ± 0.8
2.31 ± 0.9
15.2 ± 0.6%
10.52 ± 0.6%***
Total IBA-1 positive cells
217.94 ± 14.8
74.45 ± 12.3***
IBA1/BrdU positive cells
19.66 ± 3.6
4.71 ± 3.5
9.02 ± 1.2%
6.32 ± 1.8%*
Percentage of IBA-1/BrdU-positive cells outside the demyelinated region.
5 th postsurgery day
7.74 ± 0.44%
10.29 ± 0.31%
7 th postsurgery day
1.12 ± 0.01% a
7.22 ± 0.12% ***
In the CNS, myelination is accomplished by mature oligodendrocytes that arise from OPCs. During CNS development, a substantial pool of OPCs must be generated for production of mature oligodendrocytes in sufficient numbers for adequate myelination of axons. The adult CNS still contains large numbers of OPCs that differ somewhat from perinatal progenitors in their capability for motility and proliferation, yet respond to most of the same stimuli and express a similar set of phenotypic markers as their perinatal counterparts. Adult OPCs account for a large percentage of the proliferating cells in the mature CNS [7, 9] and are responsible for production of new oligodendrocytes to replace damaged cells. Newly-differentiated oligodendrocytes derived from adult OPCs, rather than pre-existing oligodendrocytes, are responsible for remyelination of axons that occurs following various types of demyelinating events [8, 10, 32–34]. Factors that influence OPC proliferation and differentiation are therefore of great importance for our understanding of both developmental myelination and myelin repair.
The NG2 proteoglycan contributes to the proliferation of OPCs during CNS development. In the NG2 null mouse, decreased OPC proliferation reduces the size of the OPC pool, leading to a delay in production of normal numbers of mature oligodendrocytes and to a corresponding delay in axon myelination . We have used lysolecithin-induced demyelination of the spinal cord to examine the possibility that ablation of NG2 also impedes repair of myelin damage in the adult CNS. Following microinjection into CNS white matter, lysolecithin replaces phospholipids and forms micelles in the membrane bilayer , rapidly inducing local myelin destruction , blood-brain barrier damage, and recruitment of macrophages and local microglial cells into the lesion site . This commonly-used demyelination model [4, 19, 35–37] has the advantage that the site and extent of the injury are well-defined and reproducible, facilitating data acquisition. In addition, lysolecithin-induced demyelination occurs as an acute event, such that all subsequent phenomena are associated with the regenerative response. This provides a useful means of separating events and mechanisms associated with the respective processes of demyelination and remyelination .
The regeneration of myelin following demyelination is a multifactorial process, due in part to the involvement of multiple cell types in the damage and repair mechanisms. In addition to neurons and OPCs, microglia, macrophages, and pericytes also contribute to these processes [38–41]. Our work shows that the NG2 proteoglycan is expressed by three cell types that invade demyelinated lesions: OPCs, pericytes, and macrophages/microglia. The differential contributions of these three cell types to the damage and repair processes, combined with differences in NG2 function in the respective cell types, are probably responsible for the complex patterns of demyelination and remyelination that we see in the global NG2 null mouse. Figure 2 shows that although the extent of initial demyelination is reduced in the NG2 null mouse, repair of this lesion nevertheless proceeds more slowly than repair of the larger lesion found in the wild type mouse. The impact of NG2 ablation on OPCs is likely confined to deficiencies seen during the repair process, since OPCs generate oligodendrocytes that carry out remyelination. Conversely, diminished involvement of macrophages/microglia probably provides the best explanation for the reduced extent of initial demyelination seen in the NG2 null mouse. However, macrophages/microglial cells also contribute to myelin repair by clearing myelin debris and by producing cytokines and growth factors that promote recruitment of OPCs and prime interactions between OPCs and axons. Thus, NG2-dependent deficits in macrophage/microglia function may also contribute to the reduced myelin repair seen in the NG2 null mouse. Similarly, it is possible that pericytes affect both myelin damage and repair. The recruitment of pericytes for revascularization of the lesion and repair of the blood brain barrier likely plays an important role in the healing process. However, vascularization also provides increased access to inflammatory cells and cytokines that contribute to myelin damage [40, 42–45]. Since many of the pericytes in lysolecithin-induced lesions are not associated with vascular endothelial cells, another consideration is the ability of pericytes to serve as mesenchymal stem cells [46, 47] with immunomodulatory properties that can promote myelin repair via their effects on the activities of inflammatory cells .
Our evidence suggests that promoting cell proliferation is a key functional role for NG2 in OPCs, pericytes, and macrophages/microglia. BrdU incorporation reveals significant reductions in mitotic index for all three cell types in demyelinated lesions in the NG2 null mouse. In the case of OPCs, this confirms a similar result obtained in our studies of developmental myelination: namely, that ablation of NG2 reduces the OPC mitotic index, with a corresponding decrease in the number of myelinating oligodendrocytes . Thus, NG2 is important for promoting the proliferation of both perinatal OPCs and adult OPCs. The BrdU results also confirm our report that ablation of NG2 diminishes pericyte proliferation during pathological retinal neovascularization, leading to decreased blood vessel formation in the retinas of NG2 null mice . This negative effect of NG2 ablation on cell proliferation may be a fairly general one, since we also observe diminished keratinocyte proliferation in the skin of newborn NG2 null mice . Our in vitro studies also support a role for NG2 in promoting cell proliferation. NG2 is able to enhance proliferation via two mechanisms: promotion of signaling by β1 integrins  and promotion of signaling by receptors for the growth factors PDGF and FGF [27, 51].
In vitro studies also indicate that NG2-dependent signaling by β1 integrins and growth factor receptors can promote cell motility as well as cell proliferation [27, 50, 52, 53]. In vivo, one indication of this effect is seen in our current studies on macrophage invasion into demyelinated lesions. BrdU tracking studies at day 5, one day after lysolecithin injection, show that 8 to 10% of the macrophages/microglia in dorsal column white matter are located outside demyelinated lesions. By 7 days post-injection in wild type mice, 90% of these peripherally-located cells have migrated into the lesion. By contrast, only 20% of extra-lesional cells have migrated into the lesion in NG2 null mice, indicative of the NG2 dependence of macrophage motility. Similar measurements were not possible in the case of OPCs or pericytes due to the rare occurrence of BrdU-labeled cells outside of demyelinated lesions.
Our finding of changes in cytokine expression following NG2 ablation may also be important in understanding changes in demyelination and remyelination in the NG2 null mouse. Although it remains to be determined whether changes in cytokine expression in the NG2 null mouse are associated with changes in macrophages as opposed to other inflammatory cell types, decreased levels of IFNγ and IL-1β coupled with increased levels of IL-4 and IL-10 suggest that NG2 ablation shifts a pro-inflammatory phenotype to an anti-inflammatory one. IFNγ provokes acute re-occurrence of demyelination in MS patients , and IL-1β is present in CNS-infiltrating myeloid cells in MS models . It therefore seems possible that decreased levels of IFNγ and IL-1β in spinal cord lesions in the NG2 null mouse (or altered activities of cells expressing these cytokines) are responsible for the reduced white matter damage seen in these mice. Moreover, decreased IL-4 production in the CNS exacerbates experimental autoimmune encephalitis, and is associated with increased infiltration of inflammatory cells , while increased IL-10 expression is associated with reduced inflammation . The possibility that NG2 null macrophages/microglia may exhibit less inflammatory properties than wild type cells is in line with the in vitro finding of a reduced inflammatory phenotype upon knockdown of NG2 in microglia . We speculate that diminished occurrence of myeloid cells in NG2 null spinal cord lesions, coupled with alterations in the intrinsic properties/functions of NG2-negative macrophages/microglial cells, can affect the progression of demyelination and remyelination in NG2 null mice.
In summary, our results demonstrate that the functions of all three of the NG2-positive cell types (OPCs, pericytes, and macrophages/microglia) associated with demyelinated lesions are compromised by the ablation of NG2. As a result of changes in multiple cell types, the respective processes of myelin damage and myelin repair are both altered in NG2 null mice. The complexity of the demyelination/remyelination phenotype in the global NG2 null mouse suggests that cell type-specific ablation of the proteoglycan will be a useful strategy for elucidating the respective contributions of NG2-positive cell types to the myelin damage and repair processes. The use of NG2 floxed mice in conjunction with appropriate Cre drivers will allow us to perform the desired NG2 ablations.
List of abbreviations
central nervous system
myelin basic protein
NG2 null mice
wild type mice
oligodendrocyte progenitor cells
Plate derived growth factor receptor
quantitative reverse transcription-polymerase chain reaction.
This work was supported by Postdoctoral Fellowship 82922 from the Craig H. Neilsen Foundation (KK) and by NIH grants PO1 HD25938 and RO1 CA95287 (WBS). We thank Dr. Michael Hefferan and Dr. Viktor Skihar for help with spinal cord surgery, and Francisco Beltran and Adriana Charbono for assistance with portions of the animal work.
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