Mice lacking Nogo-A/B, MAG and OMgp are viable and display normal CNS architecture
In order to ascertain the interactions and the relative potencies of Nogo, MAG and OMgp to inhibit axon growth, we generated single, double and triple null mutant mice for Nogo (N−/−), MAG/OMgp (MO−/−) and Nogo/MAG/OMgp (NMO−/−) respectively. The mag−/−
mice (Bartsch et al., 1995
) were bred with omgp−/−
mice (Ji et al., 2008
) and backcrossed until homozygote double mutants were generated. Progeny from mag/omgp
double knockouts were intercrossed with the nogoabtrap/trap
mice (Kim et al., 2003
; Cafferty et al., 2007a
) to produce nogotrap/trapmag−/−omgp−/−
triple knockout mice. Western blot analysis of cortical brain lysate confirms the absence of Nogo-A in single mutant mice, of MAG and OMgp in double mutant mice and of all three ligands in the triple mutant mice (). Expression of myelin basic protein (MBP) and NgR1 is similar in the mutant mice in comparison to wild type mice.
nogoabtrap/trapmag−/−omgp−/− mice have normal brain anatomy
Discrete myelin abnormalities at particular ages have been reported for each of these three mutant strains (Li et al., 1994
; Montag et al., 1994
; Bartsch, 1996
; Huang et al., 2005
; Pernet et al., 2008
), so we considered whether the double or triple mutant mice might display more pronounced abnormalities in overall CNS myelination. Regional patterns of myelination assessed by anti-MBP staining in brain and spinal cord of nogoabtrap/trap
() and nogoabtrap/trapmag−/−omgp−/−
mice (), are identical to that observed in wild type mice (). Furthermore, there is no qualitative difference in cortical or medullary spinal cord neuronal number or distribution in nogoabtrap/trap
() and nogoabtrap/trapmag−/−omgp−/−
mice (). Thus, neuronal development is remarkably normal in mice lacking all three myelin inhibitors.
Synergistic CNS myelin inhibition of neurite outgrowth by MAG/OMgp is revealed in absence of Nogo-A
CNS myelin inhibition of neurite outgrowth from CNS or PNS neurons is readily detected in tissue culture (Schwab and Thoenen, 1985
; Savio and Schwab, 1989
). To investigate the relative roles of Nogo, MAG and OMgp in this inhibition, we prepared myelin extracts from nogoabtrap/trap
. Dissociated dorsal root ganglia (DRG) neurons from adult wild type mice were cultured for 18 hours on 96-well tissue culture plates upon which 1 μg of extracted myelin from wild type mice, nogoabtrap/trap
mice and dialyzed extraction buffer (vehicle) had been adsorbed without dehydration. DRG cells grown on plates coated with vehicle extend neurites with an average total length of 192.5 ± 11.6 μm (), while cells grown on wild type myelin exhibit significantly reduced outgrowth with average total neurite length per well of 76.5 ± 13.5 μm (, *p < 0.01, ANOVA
). Myelin prepared from mag−/−omgp−/−
mice () exhibit a non-significant trend to less inhibition than wild type myelin, suggesting that these proteins may have little role in myelin inhibition. In contrast, myelin prepared from nogoabtrap/trap
mice is significantly less inhibitory for DRG neurite outgrowth in comparison to wild type (132.9 ± 1.8 μm vs. 76.5 ± 13.5 μm, **p < 0.01 ANOVA
) and mag−/−omgp−/−
myelin (132.9 ± 11.8 μm vs. 99 ± 10 μm, **p < 0.01 ANOVA
), demonstrating an independent and predominant role for Nogo in myelin inhibition.
Extracted myelin from nogoabtrap/trapmag−/−omgp−/− mice is a less potent inhibitor to DRG neurite outgrowth
Because Nogo, MAG and OMgp each interact with both NgR1 and PirB axonal receptors, we considered whether MAG and OMgp action might be uncovered in the absence of Nogo-A. Myelin prepared from nogoabtrap/trapmag−/−omgp−/− mice is significantly less inhibitory to neurite outgrowth () than wild type myelin (175.1 ± 25 μm vs. 76.5 ± 13.5 μm, # p < 0.01 ANOVA), mag−/−omgp−/− myelin (175.1 ± 25 μm vs. 99 ± 10 μm, # p < 0.01 ANOVA) or nogoabtrap/trap myelin (175.1 ± 25 μm vs. 132.9 ± 12.8 μm, # p < 0.01 ANOVA). Thus, the triple mutant preparation reveals a synergistic or ancillary role for MAG and OMgp in myelin limitation of axonal outgrowth on the Nogo-null background.
Rostral CST sprouting after dorsal hemisection in myelin inhibitor mutant mice
We sought to determine if a similar relationship between Nogo/MAG/OMgp genotype and axonal growth exists in vivo after traumatic injury. Dorsal hemisection (DhX) of the thoracic spinal cord results in interruption of many descending motor and ascending sensory spinal tracts, such that lesioned mice lose function in their hind limbs. Restoration of hind limb function depends largely on the re-establishment of axonal communication across the spinal cord injury site, either directly via long distance growth of injured fibers around or through the lesion site, or indirectly through local growth of injured and intact fibers to create new bypass circuits. We assessed the contribution of Nogo, MAG and OMgp to these various patterns of axon growth and recovery.
As a first step towards evaluating these multiple mechanisms, we measured the local growth of CST axons within the cervical enlargement at levels C5–C8, several mm rostral the lesion in mice of multiple genotypes. We labeled the CST via BDA microinjection into the right motor cortex (Supplementary Figure S1
). Uninjured intact wild type (), nogoabtrap/trap
() and nogoabtrap/trapmag−/−omgp−/−
mice () displayed normal CST fasciculation in the ventral dorsal columns, termination of fibers in grey matter and the sparse presence of fibers in the dorsolateral funiculi () in the cervical cord. There was no significant difference in the number of BDA+ fibers observed crossing the midline and entering cervical spinal grey matter on the ipsilateral side of sham lesioned wild type, nogoabtrap/trap
mice (, black bars). Six weeks after DhX, a small number of lesioned BDA+ fibers are seen crossing the midline in wild type () and mag−/−omgp−/−
() mice, however the number differs insignificantly from sham lesioned littermates (). Thus, injury may produce minor degrees of CST sprouting but the absence of MAG and OMgp does not alter this growth, paralleling the neurite outgrowth assays in the presence of myelin ().
Bilateral sprouting of BDA+ CST fibers in cervical spinal cord after dorsal hemisection at T8 in nogoabtrap/trapmag−/−omgp−/− mice
In contrast to control mice, nogoabtrap/trap
mice exhibit significantly increased numbers of BDA+ CST fibers crossing the midline after DhX in comparison to intact and lesioned wild type or mag−/−omgp−/−
mice, or intact nogoabtrap/trap
mice (, * p < 0.001, # p < 0.01, ANOVA
). Even greater degrees of BDA+ CST sprouting across the midline is observed in nogoabtrap/trapmag−/−omgp−/−
mice after DhX in comparison to intact and lesioned wild type, mag−/−omgp−/−, nogoabtrap/trap
mice and intact nogoabtrap/trapmag−/−omgp−/−
mice (, ** p < 0.001, ## p < 0.01, ANOVA
). The reproducibility of this phenotype is illustrated in Suppl. Fig. S2
. Thus, the absence of MAG/OMgp increases a Nogo-deficient post-injury axonal sprouting phenotype, and this matches the in vitro
myelin inhibition of axonal growth ().
CST regeneration after dorsal hemisection in nogoabtrap/trapmag−/−omgp−/− mice
The sprouting of CST fibers in the cervical spinal cord of nogoabtrap/trap
mice rostral to the T8 dorsal hemisection indicates that intact spinal grey and white matter are less inhibitory to axon growth. In order to test whether lesioned areas are more permissive for axon growth in nogoabtrap/trap
mice in comparison to wild type mice, we assessed the growth of BDA+ CST fibers in sagittal sections spanning a T8 dorsal hemisection (). The CST fails to grow caudal to the DhX in wild type mice (). CST axons are seen at the lesion perimeter, and most of these axons exhibit dystrophic end bulbs characteristic of abortive regeneration ( Aii
, arrows, (Silver and Miller, 2004
)). Camera Lucida reconstruction of serial sagittal sections through the lesion site illustrates the absence of BDA+ CST axons caudal to the lesion () in wild type mice after DhX, the same phenotype was observed in nogoabWT/trapmag+/−omgp+/−
mice () and mag−/−omgp−/−
mice (). Significant numbers of regenerating BDA+ CST fibers were observed growing at 1, 2, and 3 mm past the lesion site in nogoabtrap/trap
(, * p < 0.01 ANOVA
) and nogoabtrap/trapmag−/−omgp−/−
mice (, ** p < 0.01 ANOVA
) in comparison to wild type and nogoabWT/trapmag+/−omgp+/−
mice. Furthermore, nogoabtrap/trapmag−/−omgp−/−
mice displayed significantly more BDA+ CST axons after DhX at 1, 2 and 3 mm caudal to the lesion site in comparison to nogoabtrap/trap
mice (, ** p < 0.001 ANOVA
). High power photomicrographs i and ii illustrate the irregular growth patterns, characteristic (Steward et al., 2003
) of regenerated fibers. The pattern of increased growth in the absence of Nogo increased by the absence of MAG and OMgp parallels the in vitro
findings and rostral CST sprouting results.
Regeneration of BDA+ CST axons in nogoabtrap/trapmag−/−omgp−/− mice
Regenerating CST axons preferentially grow in white matter caudal to the lesion
We sought to ascertain whether regenerating fibers crossing the lesion in nogoabtrap/trapmag−/−omgp−/− mice preferentially grew in white or grey matter. We collected serial sections from a nogoabtrap/trapmag−/−omgp−/− mouse and reconstructed one side of the spinal cord using all sections with evidence of BDA+ axons (). Photomicrographs A, C, E, G are projection images of 4 sagittal sections and represent 4 regions of spinal cord depicted in schematic. The green represents for the central zone (mostly dorsal and ventral column white matter and central canal), blue for the medial zone (mostly dorsal and ventral grey matter), yellow for the medio-lateral zone (grey > white matter) and red for the lateral zone (grey < white mater). Camera Lucida reconstructions B, D, F and H represent each of the collapsed images in the projection in a distinct color. Most axons caudal to the lesion site appear in zones with a greater percentage of white matter. The percentage of BDA+ CST axons observed in white matter 2 mm caudal to the lesion site in nogoabtrap/trapmag−/−omgp−/− mice is 62.1 ± 1.4 % (n = 9, ± SEM).
Regenerating CST axons preferentially grow in white matter in nogoabtrap/trapmag−/−omgp−/− mice
To confirm that the large number of axons present at and past the lesion site () reflect regenerative growth, we immunostained sagittal sections of spinal cord from each of the 4 zones with GFAP to observe the extent of injury-induced astrocyte activation (see below). The GFAP-IR images were projected onto the Camera Lucida reconstruction of axonal growth () to demarcate the lesion site relative to regenerating axons (). GFAP-IR can be seen extending from the dorsal surface of the spinal cord to below the central canal (). Thus, reconstruction demonstrates that CST fibers regenerate extensively into white matter greater than gray matter in the caudal spinal cord of nogoabtrap/trapmag−/−omgp−/− mice.
Raphespinal regeneration after dorsal hemisection in nogoabtrap/trapmag−/−omgp−/− mice
The DhX injury damages multiple spinal tracts in addition to the CST, including the serotonergic raphepsinal tract (RST). The RST contributes significantly to locomotion (Lemon, 2008
). To determine whether the growth of raphespinal axons is differentially sensitive to the presence of NogoA/B, MAG and OMgp, we assessed the growth pattern of the RST after DhX in wild type, nogoabtrap/trap
mice. 5HT-immunoreactive (5HT-IR) RST axons originating in the brainstem descend bilaterally in the lateral columns and densely innervate both dorsal and ventral grey matter at all spinal levels. We focused our analysis on the ventral horn of the lumbar spinal cord where the RST is know to synapse on motorneurons (Mason, 2001
). 5HT-IR axons are observed to ramify densely in the L4/5 ventral horn of sham lesioned wild type and nogoabtrap/trapmag−/−omgp−/−
mice (). DhX at T8 significantly reduces the density of 5HT-IR by 75% in wild type (, * p < 0.001 ANOVA
) and mag−/−omgp−/−
mice (, * p < 0.001 ANOVA
). The reduction of caudal 5HT fibers is less pronounced in nogoabtrap/trap
mice (, * p < 0.001 ANOVA
). In obvious distinction, the density of 5HT innervation in the L4/5 ventral horn of nogoabtrap/trapmag−/−omgp−/−
mice is restored fully, being insignificantly different from sham lesioned wild type or nogoabtrap/trapmag−/−omgp−/−
control mice (). The intact RST innervation likely reflects caudal sprouting of these fibers after the injury to restore normal fiber density. As for other measures, there is no regenerative phenotype in mag−/−omgp−/−
mice, but the partial phenotype in the nogoabtrap/trap
mice is enhanced by deletion of MAG and OMgp.
Regeneration of RST axons in nogoabtrap/trapmag−/−omgp−/− mice
Locomotor recovery in nogoabtrap/trapmag−/−omgp−/− mice after SCI
The primary outcome in this study is the degree of axonal growth after spinal lesion in mice of different genotypes. However, we also scored functional outcomes by observing locomotion in the open field for each cohort. Standardizing lesion severity in experimental SCI models is crucial to correlating anatomical with functional outcomes. Therefore, we assessed lesion depth in animals that underwent dorsal hemisection (), using GFAP immunoreactvity. Wild type, nogoabtrap/trap (), mag−/−omgp−/− (), nogoabWT/trapmag+/−omgp+/− () and nogoabWT/trapmag+/−omgp+/− nogoabtrap/trapmag−/−omgp−/− mice () display equivalent induction of GFAP at the lesion site 5 weeks post lesion. The percentage of GFAP-negative spared spinal tissue in the remaining groups of animals is insignificantly different between genotypes ().
Improved locomotor recovery in mice lacking nogoA/B after DhX
Mice behavior was assessed on day 3 pre-lesion, and 6 hours, 3, 7, 14, 21 and 28-days post lesion (). All animals display flaccid hind limb paralysis 6 hours after lesion and slowly regain function over the observation period. Only nogoabtrap/trap and nogoabtrap/trapmag−/−omgp−/− mice recovered significant hind limb function in comparison to nogoabwt/trap (, Repeated Measures ANOVA p < 0.001) and nogoabwt/trapmag+/−omgp+/− control mice (, Repeated Measures ANOVA p < 0.001), mag−/−omgp−/− mice failed to recover significant function in comparison to mag+/−omgp+/− mice (). There was no significant difference in BMS scores between wild type, nogoabwt/trap, mag+/− omgp+/− and nogoabwt/trapmag+/−omgp+/− control mice after DhX (). Therefore we pooled the control groups (, WT/HT) for comparison to single, double and triple mutants. Both nogoabtrap/trap and nogoabtrap/trapmag−/−omgp−/− mice recovered significant function in comparison to controls (, Repeated Measures ANOVA, p < 0.005 and p < 0.001 respectively). Furthermore nogoabtrap/trapmag−/−omgp−/− mice recovered to a greater degree than do nogoabtrap/trap mice (Repeated Measures ANOVA, p < 0.05). There was no significant difference between mag−/−omgp−/− and control mice.
To support the robustness of this finding across injury severity, we examined a separate cohort of nogoabtrap/trapmag−/−omgp−/− and nogoabWT/trapmag+/−omgp+/− that had a more severe, near total transection lesion due to deeper cutting at the time of surgery. Such mice preserve less than 10% of tissue (). The severely injured nogoabtrap/trapmag−/−omgp−/− mice recover greater function in the open field than do the nogoabWT/trapmag+/−omgp+/− mice (, Repeated Measures ANOVA p < 0.001).
Improved locomotor recovery in mice lacking Nogo-A/B after sub-complete transection