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Measuring in vivo spinal cord injury and repair remains elusive. Using MRS we examined brainstem N-acetyl-aspartate as a surrogate for spinal cord injury in two mouse strains with different reparative phenotypes following virus-induced demyelination. SJL mice progressively demyelinate with axonal loss. FVB mice demyelinate similarly but eventually remyelinate coincident with functional recovery. Brainstem NAA levels drop in both but recover in FVB mice. Chronically infected SJL mice lost 30.5% of spinal cord axons compared to FVB mice (7.3%). In remyelination-enhancing or axon-preserving clinical trials, brainstem MRS may be a viable endpoint to represent overall spinal cord dysfunction.
New, non-invasive technologies to evaluate spinal cord injury and repair are needed. Proton magnetic resonance spectroscopy (MRS) is one ideal method; however, because the spinal cord is small and surrounded by bone, direct spectroscopy has been limited. During retrograde labeling studies of demyelinated spinal cord axons, we observed a reduction in neuron cell bodies in the brainstem labeled with fluorescent markers indicating death or dysfunction1. This led to the concept that neuron health measured at the brainstem with MRS may reflect the integrity of many ascending and descending spinal cord pathways. By following virus induced spinal cord disease in two strains of mice, we show that chronic demyelination with axonal loss and remyelination with axonal preservation result in different brainstem MRS signatures. This provides proof in principle that assessment of brainstem MRS in humans may serve as a surrogate marker of spinal cord axonal health or injury.
TMEV infection in susceptible strains of mice results in immune-mediated chronic CNS demyelination and is an excellent model for MS2. Histopathology includes focal demyelinated lesions, inflammation, axonal damage, glial scars and rarely remyelination. Infection of SJL mice results in chronic demyelination with very little remyelination, whereas infection of FVB mice results in similar extent of demyelination followed by almost complete remyelination (Figure 1). Retrograde labeling demonstrated significant injury to axons and their corresponding neurons in the brainstem in SJL mice but only limited injury in FVB mice3.
Proton MR spectroscopy (1H-MRS) is a non-invasive method that enables in vivo quantification of metabolites in the brainstem. Important MRS peaks in nervous tissue are N-acetyl aspartate (NAA), myo-inositol, creatine/phosphocreatine (Cr) and choline-containing compounds (Cho). NAA is the most abundant free amino acid in nervous tissue4. NAA concentration is considered to reflect primarily neuronal and axonal integrity because NAA is almost exclusively restricted to neurons. The MR spectral profile of purified CNS cells indicates that NAA signal amplitude is predominant in neurons. NAA signal amplitude in purified cultures of oligodendrocytes or astrocytes was 5% and 10% of the neuron signal, respectively 5. In the MRS spectrum, the dominant NAA peak occurs at 2.02 ppm, originating from 3 N-acetyl methyl group protons 4. The area under the MRS peak is proportional to the number of spins in the group and can be converted into molar concentration, after proton number normalization6. NAA decreases in neurons after injury and is a marker of integrity. A decrease of NAA concentration occurs early in disease in normal-appearing white matter of MS patients7–10. Of importance, the relative NAA concentration correlates with the neurologic disability in MS patients11. In this study, we use brainstem MRS as a surrogate marker for spinal cord neuronal integrity in strains of mice with different phenotypes of virus-induced demyelinating disease.
SJL/J (Swiss Jim Lambert) and FVB/NJ (Friend Virus B) mice (Jackson Laboratories, Bar Harbor, ME) were housed and bred in Mayo Clinic’s animal care facility. Animal protocols were approved by the Mayo Clinic Institutional Animal Care and Use Committee.
Demyelinating disease was induced in 6- to 8-week-old mice by intracerebral injection of TMEV (Theiler’s murine encephalomyelitis virus). See supplementary methods for more details on virus infection. Axon injury and loss begin three months after infection correlating with neurologic dysfunction12.
MRS was performed using a Bruker Avance 300MHz (7T) vertical bore NMR spectrometer (Bruker Biospin, Billercia, MA) equipped with mini- and micro-imaging accessories. During data acquisition, animal core temperature was maintained at 37°C by a flow of warm air. Inhalational isoflurane anesthesia 1.5–2.5 % in oxygen was delivered via nose cone. MRS data were obtained from a (2.5×2.5×2.5) mm3 voxel, placed over the brainstem (Figure 1C) and (3×3×3.5) mm3 voxel placed over the striatum as a control (Supplementary Figure 1A). Spectra were processed and analyzed with TOPSPIN, Bruker Biospin’s proprietary software. NAA was quantified by comparing the areas under NAA peaks from the brainstem with the area under the same peak in a test phantom standard with known concentration, recorded under identical conditions, as explained in detail in supplementary methods. Overlapping spectra from a healthy and infected mouse are shown (Figure 1D).
Mice were perfused and spinal cords embedded in araldite plastic (see supplementary methods for details on morphology). An Olympus Provis AX70 microscope that was fitted with a DP70 digital camera and a 60x oil-immersion objective was used to capture six sample areas of normal-appearing white matter containing a relative absence of demyelination from each cross section, according to the sampling scheme in Figure 3A. Approximately 400,000 μm2 of white matter was sampled from each mouse. Absolute myelinated axon numbers were calculated as previously reported13. Methods for brain pathology scores are described in supplementary data.
Data were compared by Student’s T test if normally distributed or by Mann-Whitney Rank sum test if non-normally distributed. More than two groups were compared by ANOVA. P<0.05 was considered significant.
MRS was collected from 3 groups of SJL and FVB mice (n=8–10): normal uninfected, 90 days post-infection and 270 days post-infection. At 90 days post-infection brainstem NAA concentrations decreased in both strains compared to uninfected controls (Figure 2A and C). At 270 days post infection, NAA concentrations in SJL mice remained low, while NAA levels recovered in FVB mice. This is consistent with histopathology and disease phenotype SJL––mice developed axon loss following demyelination12 whereas FVB mice repair1.
To characterize the temporal changes in NAA concentrations through disease in individual mice, MRS was collected from groups of SJL and FVB mice (n=15) prior to TMEV infection and at 21, 45, 90, 180 and 270 days after infection (Figure 2). Confirming our first study, NAA concentrations fell in both strains at 90 days after infection and remained low 90 days later. Again, at the 270 days post infection, NAA levels recovered close to baseline in FVB mice but remained low in SJL mice (Figure 2B and D). In a separate cohort of SJL and FVB mice at 0, 90 and 270dpi MRS was collected over the striatum as a control for the brain stem values. No differences were found in NAA concentrations in the striatum (Supplementary Figure 1B). In the same cohort of infected mice, hemispheric and brain stem MRI was performed, showing no lesions (Supplementary Figure 2A–H). Brain pathology assessment did not reveal significant difference between the two strains p=0.216 (Supplementary Figure 2I and J)
At the end of the longitudinal study, axons were counted in the normal-appearing spinal cord white matter at T6 in each mouse (Figure 3B). There were 30.5% fewer axons in SJL mice at 270dpi compared to age-matched uninfected controls (p<0.001). In contrast, there were 7.3% fewer axons in FVB mice at 270dpi, which was not different from uninfected controls (p=0.134). We found positive correlation between brain stem NAA concentrations and axon counts in both mouse strains (Figure 3C and D). For SJL mice r=0.823 (p=0.012) and for FVB mice r=0.775 (p=0.005).
Infection of susceptible strains of mice with TMEV induces inflammatory demyelination in the spinal cord 3. In SJL mice, demyelination begins several weeks after infection with minimal remyelination and progressive neurologic deficits. FVB mice are similar in ancestry to SJL mice14 but differ at the MHC H-2 locus. By three months after infection, spinal cord demyelination is equivalent in the two stains. However, three months post infection, the disease progression diverges. SJL remain demyelinated, whereas FVB mice completely remyelinate and recover neurologic function3.
Retrograde labeling at the T6 level of the spinal cord with the fluorescent tracer Fluoro-Gold 1 shows that demyelination in SJL mice is accompanied by axonal loss, detected as a decrease of fluorescent brainstem neurons. Most lesions occur at the cervical and thoracic level. Therefore, a reduction in the number of labeled brainstem cells occurs because of disturbed retrograde transport or axonal degeneration12. When compared to uninfected controls, chronically infected SJL mice show >70% reduction in retrograde-labeled brainstem cells. At 270 days after infection, the brainstem is not demyelinated by electron microscopy; thus, the changes in MRS in brainstem are a result of retrograde injury from the spinal cord
Magnetic resonance spectroscopy is widely used in models of neurologic disease. To our knowledge, this is the first study using MRS at the brainstem as a surrogate marker for axonal injury and demyelination throughout the spinal cord. Two possible explanations for the recovery of NAA levels in FVB mice are: 1) brainstem neurons are only injured—not destroyed—during demyelination and regain function as repair progresses; or 2) brainstem neurons are lost, but later replaced by precursor cells. Because disability in human MS patients is often determined primarily by spinal cord lesion load, MRS at the brainstem may predict disease progression. MRS at the brainstem may also be a viable endpoint in clinical trials designed to preserve or protect axons in the spinal cord.
Disclosure: This work was supported by grants from the NIH (NS R01 24180, NS R0132129, NS048357) and grants from the National Multiple Sclerosis Society (RG 3172 and CA 1011A8). We also wish to thank the Applebaum and Hilton Foundations for their support.
We appreciate the editorial assistance of Lea Dacy.