Manganese‐enhanced magnetic resonance imaging for in vivo assessment of damage and functional improvement following spinal cord injury in mice

In past decades, much effort has been invested in developing therapies for spinal injuries. Lack of standardization of clinical read‐out measures, however, makes direct comparison of experimental therapies difficult. Damage and therapeutic effects in vivo are routinely evaluated using rather subjective behavioral tests. Here we show that manganese‐enhanced magnetic resonance imaging (MEMRI) can be used to examine the extent of damage following spinal cord injury (SCI) in mice in vivo. Injection of MnCl2 solution into the cerebrospinal fluid leads to manganese uptake into the spinal cord. Furthermore, after injury MEMRI‐derived quantitative measures correlate closely with clinical locomotor scores. Improved locomotion due to treating the detrimental effects of SCI with an established therapy (neutralization of CD95Ligand) is reflected in an increase of manganese uptake into the injured spinal cord. Therefore, we demonstrate that MEMRI is a sensitive and objective tool for in vivo visualization and quantification of damage and functional improvement after SCI. Thus, MEMRI can serve as a reproducible surrogate measure of the clinical status of the spinal cord in mice, potentially becoming a standard approach for evaluating experimental therapies. Magn Reson Med, 2006. © 2006 Wiley‐Liss, Inc.

Traditionally, neuroanatomic connections have been primarily studied in animals using degeneration methods (1) and anterograde/retrograde tracing techniques (2). These techniques have two major disadvantages. First, animals must be sacrificed for further processing for histology precluding longitudinal studies in the same subject. Second, these techniques yield structural information of connections but exclude the possibility of functional assessment.
Mn 2ϩ is known to cause a shortening of the T 1 time of water protons in MRI leading to a strong contrast enhancement in T 1 -weighted MRI (3). Mn 2ϩ is a divalent ion with chemical properties resembling Ca 2ϩ . It is actively transported into neurons via voltage-gated Ca 2ϩ channels (4). Generally, three major applications of manganese-enhanced magnetic resonance imaging (MEMRI) have developed (5): (1) as a tissue contrast agent, (2) as a surrogate marker for neuronal cell activity, and (3) for tracing of neuronal tracts. First, after systemic MnCl 2 injection in rodents, specific uptake patterns of Mn 2ϩ with enrichment within the gray matter of the brain were described (6,7). Second, Mn 2ϩ has been successfully used as a Ca 2ϩ analog to visualize activity-dependent uptake into the rat brain (3). This has also been shown in songbirds, where an injection of MnCl 2 solution into the cortex gave rise to a selective pattern of Mn 2ϩ uptake in the brain (8). Importantly, the amount of uptake was dependent on the level of neuronal activity (9). Third, Pautler et al. were the first to exploit MEMRI for depicting neuronal connections. Injection of MnCl 2 solution into the vitreal chamber enabled visualization of the optical tract in mice (10). Consecutively, multiple other uses have been described like intracranial (i.c.) MnCl 2 injection that resulted in the enhancement of several white matter tracts of the monkey brain (11). Once inside an axon, Mn 2ϩ is transported in both antero-and retrograde direction. Transsynaptic propagation has also been observed (12).
In past years the development of MRI techniques for in vivo examination of the injured spinal cord has been accelerated. Promising approaches using functional MRI in rodents have been designed by different groups (13,14). Similarly, diffusion anisotropy MRI has been applied for quantitative assessment of recovery following spinal cord injury (SCI) in rats (15). Very recently, the potential use of MEMRI to examine the axonal connectivity after SCI has been explored (16). Nevertheless, the high intrinsic variability of these methods, although generally functional, renders them unsuitable for quantitative discrimination of minute differences in spinal cord integrity, especially in small animals like mice.
To date, no data are available on the in vivo visualization of the functional status of the mouse spinal cord using MnCl 2 . We hypothesized that an intracerebroventricular (i.c.v.) injection of MnCl 2 would lead to spinal Mn 2ϩ uptake with the amount of Mn 2ϩ uptake into the spinal cord depending on its functional status. We tested this hypoth-esis in a mouse model of SCI, in which animals were treated with a therapy (neutralizing antibodies against CD95Ligand) that is neuroprotective and leads to axonal regeneration resulting in strong functional improvement (6,17). Here, we show that MEMRI-derived parameters correlate closely with clinical scores of locomotor function. Therefore, MEMRI may serve as a sensitive in vivo method for monitoring neuronal activity and functionality within the spinal cord and, moreover, could provide an objective in vivo parameter for the evaluation of damage after SCI and treatment effects.

Spinal Cord Injury
Animal experiments were approved by the German Cancer Research Center institutional animal care and use committee and the Regierungspräsidium Karlsruhe. For SCI, MnCl 2 injection and MRI scan animals were anesthetized using isoflurane. SCI was performed essentially as described before (17). In brief, after laminectomy at the level Th 7/8 the dorsal 80% of the spinal cord were transected using fine irridectomy scissors leaving only the ventral funiculus intact. This procedure results in complete paraplegia directly after injury. Minor spontaneous recovery of hind limb function is usually observed in the consecutive weeks. In the double-blind therapy experiment, injured mice received saline solution or were treated with 50 g neutralizing antibodies directed against CD95Ligand (MFL3; Pharmingen). Antibody and saline were injected intraperitoneally (i.p.) 30 min before SCI as well as twice weekly in the following 5 weeks.

Contrast Agent Injection
For i.c.v. injections anesthetized animals were mounted onto a stereotaxic frame. A total of 0.25 L of 0.8 M MnCl 2 solution in saline was injected bilaterally into the lateral ventricles (coordinates: 1 mm caudal to the bregma, 1.5 mm lateral, 1.8 mm depth) using a microinjector (Harvard Apparatus). For cisterna magna injections, 0.5 L of 0.8 M MnCl 2 solution or 10 L of 0.5 M gadolinium-DTPA solution (Magnevist, Schering), respectively, was injected using a 27-g needle attached to a microsyringe as described (18). The MnCl 2 dose for intravenous (i.v.) application into the tail vein was 0.2 mmol/kg and 1 mmol/kg for subcutaneous (s.c.) administration.

Study Groups
The study was divided into several parts. In the first part, the time course experiment of Mn 2ϩ uptake into the spinal cord, we performed an i.c.v. injection of MnCl 2 solution in one animal and measured the contrast enhancement in the spinal cord 0, 1, 2, 4, 8, 12, 24, 72, and 120 h afterward. To evaluate alternative application routes, MnCl 2 solution was administered either i.v., s.c., or into the cisterna magna (n ϭ 2 per group). Imaging was performed 8 to 24 h after injection. In the second part, the comparison of contrast enhancement in the spinal cords of injured and uninjured mice, animals subjected to SCI (n ϭ 10) and uninjured controls (n ϭ 5) received MnCl 2 injections 5 weeks after SCI. MRI followed 60 h later. In the third part, the therapy experiment, MnCl 2 solution was injected into antibody-treated and control mice (n ϭ 6 per group) at 4 h and 5 weeks after SCI. Imaging was always performed 60 h after injection. To examine the integrity of the cerebrospinal fluid (CSF) circulation system, gadolinium-DTPA was injected into the cisterna magna of uninjured (n ϭ 2) and injured mice at 5 weeks after SCI (n ϭ 6). MRI followed at multiple time points between 5 min and 6 h after injection.

Behavioral Testing
All tests were performed in a double-blind manner. Spinal cord-injured mice were tested in the BBB locomotor and grid walk tests at 1, 2, 3, 4, and 5 weeks after injury. The BBB test was additionally performed after 1 day. All behavioral tests were recorded with a video camera. For assessing the overall locomotor performance the BBB locomotor rating scale was used with slight modifications (17,19). The BBB scale is a standard measure for analyzing the open field locomotor performance of rats after SCI and grades the functional status of their limbs based on different aspects like joint movement, paw placement, weight support, and coordination. Complete paraplegia results in a score of 0, while normally functional uninjured animals receive 21 points (19). Deficits in descending motor control were examined using the grid walk test (17,20). In this task the animals must traverse a horizontal ladder-like grid that is elevated above the ground. To cross this runway animals must place their limbs accurately on the irregularly spaced bars. The numbers of foot placement mistakes for 10 steps of each hind limb are counted and summed up. Uninjured mice usually make none or very few mistakes, whereas paraplegic animals are unable to step and therefore receive 20 error points.

MRI
MRI experiments were performed on a clinical 1.5-T scanner (Siemens Symphony, Erlangen, Germany) with a dedicated custom-made animal volume resonator using a 3D-FLASH imaging pulse sequence with the following parameters. For the Mn 2ϩ uptake time series experiment the parameters were as follows: TR ϭ 35.0 ms, TE

Data Processing and Statistics
Images were evaluated using the scanner software package (Syngo, Siemens). The spinal cord was outlined on axial slices and the mean signal was calculated. A second region of interest was placed outside the animal contours for noise measurement. Mean signal to noise ratio (SNR) and standard deviation (SD) were calculated for each slice. For the therapy experiment postprocessing was performed by an observer blinded to the treatment. SNR in the most proximal slice (Fig. 1c, position 3) was set to 100% and SNRs in consecutive slices were scaled accordingly expressing relative SNR (%). The area under the curve (AUC) was calculated using the scanner software package (Syngo, Siemens). The increase of Mn 2ϩ uptake depending on the position was assessed by linear regression within the two groups. Correlations between MEMRI and behavioral tests were determined using Kendall's . BBB and grid walk scores in antibody-treated mice were compared to saline controls using the Wilcoxon rank sum test.

Intracerebroventricular MnCl 2 Injection Leads to Spinal Cord Enhancement
To test the suitability of an i.c.v. MnCl 2 solution injection for spinal cord imaging we measured the time course of Mn 2ϩ enhancement after i.c.v. injection at various positions within the spinal cord. In the early time course, strong contrast enhancement was observed in the motor cortex, along the ventricle walls and in both the ventral and the dorsal parts of the cervical spinal cord (Fig. 1a). Additionally, rapid transport over the CSF was noted with uptake both via the perispinal CSF and via the CSF within the central channel (Fig. 1b). Initially, Mn 2ϩ was taken up into the spinal tissue directly adjacent to the CSF but later the spinal cord showed homogenous Mn 2ϩ distribution (Fig. 1c). The SNR within the spinal cord rose quickly in the first 24 h after injection. Afterward, a plateau phase was reached that lasted another 48 h followed by a slow Mn 2ϩ washout (Fig. 1d). This plateau reached different levels along the spinal cord with a clear increase of SNR of two to three times baseline. Residual elevated signal intensity was present up to 3 weeks after injection, after which the SNR in the spinal cord returned to baseline. Direct injection of MnCl 2 solution into the cisterna magna gave rise to a rapid increase of signal primarily along the spinal cord, suggesting a direct distribution of Mn 2ϩ via the CSF (Fig. 2). Also, no uptake in the motor cortex was noted.
Concomitantly, we also tested the suitability of less invasive modes of MnCl 2 application. Systemic administration similarly leads to signal enhancement in the brain (6,21). Mn 2ϩ enters the brain primarily via the choroid plexus, from where it is distributed through the CSF into the parenchyma (6). Thus, we examined Mn 2ϩ uptake into the spinal cord after i.v. and s.c. application. The signal intensity within the spinal cord increased slightly; however, it was strongly reduced in comparison to the i.c.v. administration (data not shown), excluding such alternative routes of administration for examining the spinal cord.

Mn 2ϩ Signal Enhancement Is Reduced after SCI
After determining Mn 2ϩ enhancement dynamics we tested the effect of SCI on Mn 2ϩ uptake. We transected the dorsal 80% of the spinal cord at the level Th7/8, which led to paraplegia. MnCl 2 solution was injected 5 weeks after SCI and imaging was performed 60 h later since at that time point a homogeneous Mn 2ϩ uptake at the lesion level could be expected (Fig. 1d). Contrast enhancement in the spinal cord is shown for representative mice without (Fig.  3a) and with injury (Fig. 3b) in the sagittal plane. A clear interruption of contrast enhancement distal to the lesion in the injured mouse can be seen as shown in more detail on the axial slices (Fig. 3c). To quantify functional impairment we determined SNR within the spinal cord on MRI images measured perpendicular to the spinal cord surrounding the lesion site (Fig. 3d). Uninjured animals displayed a homogeneous SNR throughout the spinal cord (blue curve in Fig. 3d); after MnCl 2 injection a marked increase in SNR was noted (green). Rostral to the lesion, injured mice (red) showed a SNR comparable to noninjured animals. Albeit moving further distal toward the lesion epicenter, the SNR gradually dropped, approaching background levels at the lesion site and beyond. Thus, there is an injury-dependent reduction of Mn 2ϩ uptake following SCI.
To examine whether the reduction of Mn 2ϩ uptake is due to a compromised CSF circulation, we injected gadolinium-DTPA into the cisterna magna of noninjured and SCI mice. Gadolinium-DTPA is a contrast agent that is mainly retained within the CSF (22,23). In contrast to Mn 2ϩ , gadolinium-DTPA was in all cases detected within the CSF far beyond the site of the lesion, surrounding the lumbar spinal cord (Fig. 4). This excludes the possibility that the decreased Mn 2ϩ uptake in injured mice is due to a compromised accessibility of the CSF to the spinal region caudal to the lesion.

Mn 2ϩ Uptake Correlates with Functional Recovery Following SCI
To test function-dependent Mn 2ϩ uptake we performed MRI on mice treated with neutralizing antibodies directed against CD95Ligand and their saline-treated control counterparts. We have recently shown that this therapy protects neurons and oligodendrocytes from apoptotic cell death leading to enhanced axonal regeneration and improved functional recovery following SCI (17). All animals were clinically tested once weekly using the BBB score (19) and a grid walk test (20). In saline-treated animals minor signs , the colliculus superior (c), and the pituitary (d) as described previously (22). No significant Mn 2ϩ uptake in the motor cortex was observed.
of spontaneous locomotor function recovery were observed as shown before, whereas the anti-CD95Ligand antibody-treated mice improved significantly (Fig. 5) (17).
To evaluate therapy-induced changes in spinal Mn 2ϩ uptake we performed the MEMRI experiment as described for the injured untreated animals. However, MnCl 2 solution was injected twice: at first at 4 h and for the second time at 5 weeks after SCI. Images were acquired 60 h later, and the SNR along the spinal cord was measured (Fig. 6a). We calculated the AUC for both time points and tested the value of the change in AUC (⌬AUC) as a measure of clinical improvement. In saline-treated control animals Mn 2ϩ uptake did not significantly increase with time (linear regression analysis of relative SNR (%) on the distance to the lesion epicenter: P ϭ 0.018, slope Ϫ0.269, 95% confidence interval: Ϫ0.048 to Ϫ0.49, n ϭ 6). The relative SNR (%) at 3 days and at 5 weeks after injury in a representative control animal is depicted in Fig. 6b together with the axial slices of the spinal cord of this mouse. In contrast, the anti-CD95Ligand antibody-treated animals showed a significant increase of ⌬AUC (linear regression analysis: P ϭ 0.0005, slope 1.150, 95% confidence interval: 0.831 to 1.470, n ϭ 6). In Fig. 6c the relative SNR (%) at 3 days and at 5 weeks after injury is shown for a representative treated animal. Importantly, at the early time point after SCI the MRI images of both groups were comparable. A gradual decrease of contrast enhancement along the rostrocaudal axis of the spinal cord was observed, reaching baseline levels at the lesion site, analogous to the curve of the injured untreated animals (compare Fig. 3). At the late time point, however, the images of treated and control mice differed strikingly. While in control animals the distribution of the Mn 2ϩ signal had not changed in comparison to the early measurement, the contrast enhancement pattern was strongly changed in treated mice (Fig. 6c). Around and especially caudal to the lesion site the Mn 2ϩ  uptake was significantly increased, indicating preservation of active functional neurons at the level of injury and beyond. Yet, the SNR at and caudal to the lesion site never reached uninjured levels, demonstrating residual damage despite treatment.
The changes in ⌬AUC between the early and late time points are plotted against the final behavioral test scores for both treated and control animals (Fig. 7). For testing the correlation of MEMRI with the clinical scores we ranked the 12 animals based on ⌬AUC as well as on their behavioral test scores assuming that preservation of functional neurons is the cause for both increased spinal Mn 2ϩ uptake and improved motor function. A highly significant correlation between the two rankings was found (Kendall's ϭ 0.9394, P ϭ 0.000005). Furthermore, the antibody-and saline-treated groups differed significantly with regard to MEMRI as well as BBB and grid walk ranks (Wilcoxon rank sum test; P ϭ 0.0011). In conclusion, MEMRI-derived parameters correlate closely with the clinical status as evaluated using locomotor tests.

DISCUSSION
The main problem in SCI is the interruption of neuronal connections and consecutive loss of neuronal tissue. Despite the general lack of optimism for functional recovery after SCI, in the past decade animal studies brought forward evidence for neuroprotection, axonal regeneration, and consequent regain of locomotion and of some primitive forms of sensation after experimental therapy (24,25). This has led to a great debate about the type and quality of evidence needed to select truly promising candidate therapies. In most animal studies the reported therapeutical success is based upon histologic evidence and/or assessment of the overall locomotor activity (e.g., BBB score). Major disadvantages of these methods are the need to sacrifice the animals, the great subjectivity, and the subsequent lack of standardization among the different laboratories. Here we developed an in vivo method for the evaluation of spinal cord function using MEMRI. This method has the important advantage of enabling a standardized and more objective follow-up of therapies over time.
The use of MEMRI for the functional assessment after SCI relies on the capacity of Mn 2ϩ to mimic Ca 2ϩ . Mn 2ϩ is predominantly taken up by active neurons and less by the disconnected "electrically silent" ones. The rate of axonal Mn 2ϩ transport described within the brain varies from 1.1 to 6.0 mm/h depending on injection volume and functional status of the neuronal tissue (26). Signal propagation in our experiment was much faster than that calculated for axonal transport. Moreover, experiments have shown that a focal MnCl 2 injection into the motor cortex leads to uptake in the corticospinal tract with strong signal decay in the more distal regions of the corticospinal tract without detectable contrast enhancement beyond the midbrain level (26). This indicates that in our setting visualization of the spinal cord using MEMRI is based on transport of Mn 2ϩ via the CSF with local, activity-dependent uptake into the spinal cord. This hypothesis is strengthened by the experiment, in which MnCl 2 was injected directly into the cisterna magna. Here, we did not observe uptake in the motor cortex excluding a substantial role for active axonal Mn 2ϩ transport via the corticospinal tract as a main route of contrast enhancement of the spinal cord. Instead, a strong contrast enhancement throughout the spinal cord resembling the contrast enhancement after an i.c.v. injection was evident. These findings are in accordance with a recent paper investigating i.p. application of MnCl 2 , showing uptake only in selected areas of the brain (27). Altogether, our results indicate that MEMRI of the spinal cord after i.c.v. injection is primarily based on the CSF transport of Mn 2ϩ and local activity-dependent uptake.
Injury to the spinal cord, especially in the transection model used here, may affect the integrity of CSF circulation (28). Thus, it might be hypothesized that the nearbackground levels of Mn 2ϩ uptake caudal to the lesion site found in mice directly after injury or in untreated mice 5 weeks after injury could be due to a block in CSF circulation. However, it is unlikely that the anti-CD95Ligand therapy acts on this level. Nevertheless, to exclude the possibility that our method merely depicts disruption of the CSF system, we injected gadolinium-DTPA into the cisterna magna. In all injured and uninjured control mice the contrast agent passed beyond the level of the lesion site into the lumbar spinal cord to a comparable extent. These experiments indicate that transecting the dorsal 80% of the spinal cord-although inducing local damage to the CSF compartment-does not lead to a complete disruption of the CSF circulation. Thus, the specific contrast enhancement and lack of uptake caudal to the lesion site cannot be attributed to impeded CSF circulation.
A recent publication describes Mn 2ϩ uptake in the hemi-sected spinal cord of rats after a local injection of MnCl 2 solution into the white matter of the spinal cord (16). The authors discuss their findings exclusively in terms of axonal transport but their images reveal an even stronger Mn 2ϩ uptake in the gray matter of the spinal cord than in the surrounding white matter, which indicates that in their experiments primary uptake by functional gray matter, as described in our setting, must also play a strong role. Moreover, in our experiments injection-induced trauma is minimized because an invasive and technically challenging focal injection into the white matter of the spinal cord requiring an additional laminectomy is not necessary. This is crucial for therapy monitoring since additional trauma to the spinal cord makes it more difficult to objectively determine therapy efficacy. Finally, the major advantage of our MEMRI approach is the possibility of quantitative assessment of damage and recovery.
An issue using Mn 2ϩ for in vivo imaging is its neurotoxicity (29). In contrast to focal injections into the brain parenchyma, an injection of MnCl 2 into the CSF has the advantage of rapid dilution of the toxic agent. Furthermore, in the experiments described here we stayed well within the limits of the known nontoxic dose (30). Consequently, no alterations of vital or motor functions could be observed following i.c.v. MnCl 2 injection. Symptoms of manganism that resemble disorders described as extrapyramidal motor system dysfunction, which are typical for chronic Mn 2ϩ exposure, did not develop (31).

CONCLUSION
We have devised a fast and safe in vivo method for visualization of the spinal cord and quantitative assessment of its functional status. Moreover, we demonstrated that MEMRI could substitute for behavioral tests or at least add to a more objective assessment of the spinal cord's functional status following SCI. This method could become a standard measurement for comparison of different therapies in different laboratories. MEMRI provides a surrogate read-out for damage and functional recovery after SCI. Finally, application of this method could be expanded to the study of other pathologies both in the CNS and in the PNS.