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Imaging corticospinal tract connectivity in injured rat spinal cord using manganese-enhanced MRI

Imaging corticospinal tract connectivity in injured rat spinal cord using manganese-enhanced MRI Background: Manganese-enhanced MRI (MEI) offers a novel neuroimaging modality to trace corticospinal tract (CST) in live animals. This paper expands this capability further and tests the utility of MEI to image axonal fiber connectivity in CST of injured spinal cord (SC). Methods: A rat was injured at the thoracic T4 level of the SC. The CST was labeled with manganese (Mn) injected intracortically at two weeks post injury. Next day, the injured SC was imaged using MEI and diffusion tensor imaging (DTI) modalities. Results: In vivo MEI data obtained from cervical SC confirmed that CST was successfully labeled with Mn. Ex vivo MEI data obtained from excised SC depicted Mn labeling of the CST in SC sections caudal to the lesion, which meant that Mn was transported through the injury, possibly mediated by viable CST fibers present at the injury site. Examining the ex vivo data from the injury epicenter closely revealed a thin strip of signal enhancement located ventrally between the dorsal horns. This enhancement was presumably associated with the Mn accumulation in these intact fibers projecting caudally as part of the CST. Additional measurements with DTI supported this view. Conclusion: Combining these preliminary results collectively demonstrated the feasibility of imaging fiber connectivity in experimentally injured SC using MEI. This approach may play important role in future investigations aimed at understanding the neuroplasticity in experimental SCI research. motor neurons in the SC [1]. Because, many different Background Spinal cord injury (SCI) disrupts the functional integrity types of movements are controlled through the fibers of of neuronal circuits in the ascending and descending the CST, understanding how this tract adapts to SCI is the pathways in spinal cord (SC), thereby compromising the focus of current experimental efforts [2,3]. A number of voluntary motor control of muscles below the site of the methods have been developed to anatomically trace the injury and the conduction of sensory signals from distal CST fibers using different neuronal tracers and tract trac- inputs. In SCI research, a particular pathway of interest is ing procedures [4,5]. However, these conventional meth- the corticospinal tract (CST) as it is the major long ods require elaborate tissue analysis based on histology descending output connecting the cerebral cortex with the and immunohistochemistry, and therefore preclude Page 1 of 8 (page number not for citation purposes) BMC Medical Imaging 2006, 6:15 http://www.biomedcentral.com/1471-2342/6/15 repeated measurements from the same animal. One strat- mal to confirm that the CST labeling in the SC was suc- egy to reduce the number of post mortem procedure is the cessful, and on excised cords to obtain high resolution use of a transgenic animal model that has recently been MEI data around the lesion. Here, we briefly summarize engineered to specifically and completely label the CST these procedures and give the details of the individual fibers by yellow fluorescent protein [6]. Although, this components critical to the current work. novel development simplifies the work and accelerates Spinal Cord Injury (SCI) the assessment of the regeneration and remodeling of the neuronal connections in the injured SC, it still requires Image artifacts from heart motion obscure the 3D visuali- invasive post mortem examination, making it unfit for zation of the SCI at levels below the thoracic T4 level dynamic imaging of live preparations. when the signal is acquired using volume coil. Therefore, SCI was induced at the T4 level to minimize these artifacts As an alternative method, we have shown that manga- by following the injury protocol described elsewhere [9]. nese-enhanced magnetic resonance imaging (MEI) can Briefly, the rat was anesthetized using spontaneous inha- offer new possibilities in probing the SC in live animals lation of 4% isoflurane for induction and maintained on [7,8]. MEI is an experimental technique that uses the par- a mixture of 1.5% isoflurane, 30% oxygen, and air deliv- 2+ amagnetic manganese ion (Mn ) as a contrast agent. ered through a nose mask. A rectangular area was shaved With an ionic radius similar to that of calcium-ion, Mn on the back and an incision was made to expose the pos- can enter cells through the voltage-gated calcium chan- terior elements of the spine. Then, a rongeur was used to nels, and then remain confined to the intracellular com- perform a laminectomy at T4 to expose the spinal cord, partment of intact axons. After administering Mn to the but to leave the dura intact. After stabilizing the SC with biological system, uptake and transport in the tissue of two forceps attached to the rostral T3 and caudal T5 verte- interest can be monitored remotely using in vivo magnetic bral bodies, the laminectomized section was positioned resonance imaging protocols sensitive to Mn-induced under the impactor tip of the injury device [9]. Contusion altered relaxation characteristics. Applying Mn directly injury was produced by using a rectangular (1 mm × 2 into the SC just rostral to a hemisectioned SC, we have mm) injury tip with velocity of 1.5 m/s and contact dura- demonstrated that MEI can potentially play an important tion of 80 ms. The deformation depth was set to 0.5 mm role in mapping viable neuronal tissue at and below the for producing mild partial injury with good prognosis for site of injury because functional tissue on the uninjured behavioral improvement. Next, the skin was closed and side takes up Mn and hence is delineated on the MEI [7]. the animal was placed in a heated cage to maintain the Moreover, after intracortical administration of Mn into body temperature while recovering. the motor areas of the brain and stimulating the cortex electrically, we have produced robust and detectable Intracortical Mn delivery anterograde Mn labeling of the CST from cortex caudally Intracortical delivery of Mn was achieved as described in to the thoracic SC levels [8]. The aim of the current study [8]. On post injury day 14, the injured rat was anesthe- is determine whether MEI can detect CST connectivity in tized again using ketamine hydrochloride delivered intra- partially injured SC. We used a rat animal model of mild muscularly at an initial dose of 150 mg/kg, followed by thoracic SCI. This model produces an incomplete injury additional injections at doses of 5–20 mg/kg, as needed. with functionally intact CST fibers remaining at the injury The head of the anesthetized rat was fixed in a stereotaxic site [9]. We now demonstrate that these fibers provide an frame (Kopf Instruments, Tujunga, CA). A midline inci- environment for transporting Mn caudal to the lesion. sion was made on the scalp from approximately 2.5 mm Visualization of the Mn-labeled tract below the lesion rostral to 7.5 mm caudal to the bregma, and the skin was using MEI therefore provides a method to detect the fibers retracted with hemostats. Bilateral 1.0 mm diameter burr projecting through the injured section and hence fiber holes were drilled into the skull 1.5 mm rostral to the connectivity in injured SC. bregma and 2.0 mm lateral to the midline using a 1 mm diameter trephine bit attached to a dental drill. An addi- Methods tional craniotomy was performed on one side of the skull The experiments were conducted on one Sprague-Dawley at a location 6.5 mm caudal to the bregma and 2.0 mm rat (~300 g) under a protocol approved by the University lateral to the midline. Through this opening, a 2.0 mm of Kansas Medical Center Institutional Animal Care and diameter titanium screw was inserted until it rested on the Use Committee. We followed established procedures dura. This screw served as the reference electrode for the described previously for the surgery, SCI, Mn delivery and electrical stimulation. MEI scans [8-10]. After SCI, the rat was left to recover for two weeks. On post injury day 14, Mn was delivered intra- Using 1 M MnCl in a 1 μL Hamilton syringe with a cortically and the motor cortex was stimulated electrically. tapered, graduated micropipette tip, a direct stereotaxical The next day, MEI scans were performed on the living ani- injection was made to deliver a total of 0.2 μL of this solu- Page 2 of 8 (page number not for citation purposes) BMC Medical Imaging 2006, 6:15 http://www.biomedcentral.com/1471-2342/6/15 tion focally at 0.5 mm below the surface of the cortex bilities in visualizing the Mn-labeled CST in addition to through the center of the first burr hole. The solution was the 3D GE imaging. injected slowly over a period of five minutes. To prevent backflow, the pipette was left in place for another five The MRI scans were performed 24 hr after the Mn-delivery minutes prior to withdrawal. This procedure was repeated and electrical stimulation. The rat was anesthetized using for the contra lateral motor area through the second burr spontaneous inhalation of 4% isoflurane for induction, hole. followed by a mixture of 1.5% isoflurane, 30% oxygen, and air delivered through a nose mask. The head was sta- Cortical stimulation bilized on a Plexiglas holder and positioned in a 6 cm Following the Mn delivery, electrical stimulation of the inner diameter volume coil for MRI scanning on a 9.4 T cortex was achieved with a 1 mm diameter stainless steel horizontal Varian scanner (Varian Inc., Palo Alto, CA). electrode and the titanium screw serving as the reference. While in the scanner, the physiological condition of the The electrode was connected to a DS7 Digitimer constant rat was monitored using ECG, respiratory and tempera- current stimulator (Digitimer Ltd., Hertfordshire, Eng- ture probes connected to a MR-compatible small animal land) and lowered through the first burr hole on the same monitoring and gating system (Model 1025, SA Instru- side as the screw until it touched to dura. A Grass S48 ments, Inc., Stony Brook, NY). The body temperature was stimulator (Grass Medical Instruments, Quincy, MA) gen- maintained at 37°C by circulating warm air with 40 % erated the stimulus pulse sequence used to trigger the con- humidity using a 5 cm diameter plastic tube fitted to the stant current stimulator. Trains of biphasic stimuli (each back door of the magnet bore. phase 0.2 ms, negative first) were applied at 100 Hz for an "on" period of 5 seconds followed by an "off" period of 5 After confirming the placement of the animal in the mag- seconds. The current was adjusted until an evoked visible net's isocenter with scout images, T -weighted volumetric motor response was produced in the forelimb, hindlimb images covering the brain and SC at the cervical and tho- or tail, and applied for 90 minutes. If the strength of the racic levels were acquired using a 3D GE sequence (T /T R E evoked movement declined noticeably, frequency and = 45/4 ms and flip angle (FA) = 45°. The data were sam- current values were increased to maintain a constant pled on a matrix = 128 × 128 × 64 ranging over a volume , and processed and interpolated to motor response. These procedures were then repeated for of 70 × 32 × 32 mm the opposite cortex through a second burr hole. Immedi- 256 × 256 × 128 pixels for the final display. Maximum ately after stimulation, the electrodes were removed, and intensity projections were generated to delineate the Mn the skin was sutured tightly. The animal was then left to enhancement in the CST over a desired thickness in the recover in its cage. sagittal plane. Then, a sagittal IR-MEI was acquired (T /T / R E T = 2000/12/550 ms, field-of-view (FOV) = 70 × 32 mm , Magnetic resonance imaging image matrix = 256 × 128, slice thickness = 2 mm and The paramagnetic nature of Mn changes the MR proper- NEX = 4). Finally, axial IR-MEI was acquired (T /T /T = R E I ties of the tissue where it is accumulated. In particular, sig- 2000/17/550 ms, field-of-view (FOV) = 22 × 22 mm , nals from these regions are enhanced on conventional T1- image matrix = 128 × 128, slice thickness = 2 mm and weighted spin echo (SE) images. The CST in rat SC is ana- NEX = 4). The rat was then removed from the scanner, tomically located in the ventral-most part of the dorsal euthanized using cardiac puncture and the vertebral body funiculus of the SC, i.e., near the central canal between the was dissected from the animal. The excised sample with dorsal horns of the gray matter (GM). Because of this top- intact spine was scanned ex vivo at room temperature ological arrangement, Mn-labeled CST becomes difficult using an inductively coupled surface coil centered at the to differentiate from the GM on the SE image since both injury epicenter [10,12]. High resolution multi-slice SE structures exhibit similar intensity [8]. The use of the 3D images were acquired in sagittal and axial planes (sagittal gradient-echo (GE) sequence with short repetition time parameters: T /T = 2500/12 ms, field-of-view = 32 × 10 R E however overcomes this limitation and produces robust mm , image matrix = 256 × 128, slice thickness = 0.5 mm and detectible Mn-labeled CST in SC. More recently, and NEX = 2; axial parameters: T /T = 2500/12 ms, field- R E inversion recovery SE (IR-SE) acquisitions were shown to of-view = 10 × 10 mm , image matrix = 128 × 128, slice offer better sensitivity to Mn in neuronal tissue [11]. Pre- thickness = 2 mm and NEX = 2). Finally, axial IR-MEI of viously, we have used IR-SE imaging to demonstrate the excised spine and SC were acquired (T /T /T = 2000/ R E I quantitatively that the T1-relaxation times of the GM and 15/550 ms, field-of-view (FOV) = 10 × 10 mm , image white matter (WM) are indeed slightly different in rat SC matrix = 128 × 128, slice thickness = 2 mm and NEX = 4). [10]. Based on the promise that IR-SE provides richer con- trast enhancement and our experience with this sequence, Results and discussion we also performed IR-SE imaging to demonstrate its capa- The CST in rat runs caudally from cortex through internal capsule, cerebral peduncle, longitudinal pontine fascicu- Page 3 of 8 (page number not for citation purposes) BMC Medical Imaging 2006, 6:15 http://www.biomedcentral.com/1471-2342/6/15 labeled CST centered within the SC and lesion as deline- ated by a single patch of signal hypointensity. Figure 2b shows the transverse section of the cervical SC from the imaging plane marked with a thin blue rectangle on the sagittal image. This image shows a nearly circular region of signal hyperintensity located centrally within the SC. This bright region with a well-defined boundary repre- sents the CST labeled with Mn. The corresponding data in Figs. 2c and 2d were acquired with IR-MEI and show the capability of this approach to produce in vivo signal con- trast delineating the CST. The lesion was however difficult to visualize on the IR-MEI in sagittal view. Nevertheless, Th system and projection of way Figure 1 ree-dimensional visualiz the atiocorticospinal tract n of the rat central n (CST) path- ervous Three-dimensional visualization of the rat central nervous these volumetric data confirmed that the above experi- system and projection of the corticospinal tract (CST) path- mental procedures were successful in terms of labeling the way. The data were acquired using 3D GE-MEI after 24 h of CST with Mn in live animal. intracortical injection of Mn and electrical stimulation of the motor cortex. The red color denotes the Mn-enhancement, After this confirmation, the experiment was continued labeling the CST in brain and spinal cord. with scans on the excised cord to get high resolution data. The resulting ex vivo images from this effort are shown in Fig. 3. The sagittal views in Figs. 3a and 3b show the injury lus, pyramid, pyramidal decussation, and descends in the and its extent along the SC. The anatomical images from dorsal fasciculus of the SC [13]. Figure 1 visualizes the the selected axial planes (blue rectangles in Fig. 3b) shown spatial projection of this tract in relation to the overall in Figs. 3c,d, and 3e depict the injured tissue morphology central nervous system by volume rendering of the in greater detail at the epicenter as well as the normal cord acquired GE-MEI data in 3D. The Mn-enhancement in the at sections rostral and caudal to the injury site. Figure 4 figure is represented in red. Figure 2 depicts the Mn- shows the IR-MEIs acquired from the same axial slice posi- enhancement in 2D views. The image in Fig. 2a shows a tions. The GM on these images appears relatively darker single sagittal slice depicting the Mn injection site, Mn- compared to the WM in normal sections of the cord. In In vivo pl Figure 2 anes visualization of the rat central nervous system and cross sectional views of the CST pathway in the sagittal and axial In vivo visualization of the rat central nervous system and cross sectional views of the CST pathway in the sagittal and axial planes. This data were acquired using 3D GE-MEI (a and b) and IR-MEI (c and d). The arrow labeled "SCI" points to the lesion at the T4 level. Thin-rectangles overlaid on the sagittal images represent the slice orientation for the axial images. Arrowhead in a denotes the site of the Mn injection in the brain, where the signal hypointensity is due to the presence of high local concen- tration of Mn. SI – primary somatosensory cortex, ic – internal capsule, thal – thalamus, cp – cerebral peduncle and py – pyramidal tract. Page 4 of 8 (page number not for citation purposes) BMC Medical Imaging 2006, 6:15 http://www.biomedcentral.com/1471-2342/6/15 a (right) Figure 3 , b) Ex vivo spin echo ima direction in sagittal planes ges showing the injury (arrow head) in an excised SC but intact spine from rostral (left) to caudal a, b) Ex vivo spin echo images showing the injury (arrow head) in an excised SC but intact spine from rostral (left) to caudal (right) direction in sagittal planes. c, d, e) Ex vivo anatomical axial spin echo images of the spinal cord from the three slice loca- tions (c – rostral to injury, d – injury epicenter and e – caudal to injury) depicted by the thin-rectangles overlaid on the sagittal image in b. Fig. 4a, the pattern of signal enhancement rostral to the situated right posterior to the central canal between the injury can be seen as confined spatially to the ventral- dorsal roots. Signal enhancement is also seen in Fig. 4c at most part of the dorsal funiculus between the dorsal the expected CST location caudal to the injury. Interpreta- horns of the GM in sections rostral and caudal the injury. tion of these results requires consideration of possible This enhanced region overlaps exactly with the expected mechanisms that might facilitate trans-injury propagation anatomical location of the CST in rat SC [8,13]. Careful of the Mn-dependent contrast. Likely mechanisms include examination of the image from the injury epicenter (Fig. axonal transport and extracellular diffusion of Mn. If the 4b) reveals a thin strip of signal enhancement, which is transport were by purely extracellular diffusion, we would E Figure 4 x vivo IR-MEIs from the same slice orientations as those images in Figs. 3-c, d and e Ex vivo IR-MEIs from the same slice orientations as those images in Figs. 3-c, d and e. a) Mn-enhanced CST rostral to the injury. b) Mn-enhancement at the epicenter of the injury. c) Mn-labelling of the CST caudal to the injury. The partial signal enhance- ment, depicted by the arrowhead in b, is likely to represent a portion of the CST that is populated with intact fibers. By contin- uously projecting through the injury site, these intact fibers transport Mn from rostral to caudal sections, as indicated by the presence of focal signal enhancement in c. Page 5 of 8 (page number not for citation purposes) BMC Medical Imaging 2006, 6:15 http://www.biomedcentral.com/1471-2342/6/15 Table 1: Measurements from Mn-labeled regions, above and below the lesion, and at the epicenter of the injury. Above the lesion Epicenter Below the lesion -3 2 Trace/3 (× 10 mm /s) 0.69 ± 0.05 0.59 ± 0.07 0.64 ± 0.06 FA 0.88 ± 0.08 0.79 ± 0.13 0.85 ± 0.10 Trace/3: average of the three principal eigenvalues of the diffusion tensor. FA: fractional anisotropy index. The statistics are given as mean ± standard deviation. expect that the axial extent of the enhancement region properties measured within this region would match with would expand gradually from rostral to caudal, perhaps those obtained from above and below the lesion. Process- eventually including the complete cord. However, we ing the acquired DTI data, pixel-by-pixel, produced esti- observed enhancement only in the location expected to be mates for the mean water diffusivity, diffusion anisotropy occupied by the fibers leading from the CST to distal por- and diffusion direction as respectively represented by the tions of the cord, even in the images obtained caudal to Trace/3 (average of the diffusion tensor eigenvalues) and the injury. Since the Mn enhancement remains closely fractional anisotropy (FA) parameters and principal confined to the location of the fibers into which it was eigenvector [15]. The results from these computations are originally administered, we speculate that this indicates a given in Table 1 and in Fig. 5. The quantitative Trace/3 and high level of axonal integrity since if the axons are com- FA data in each row of the table as well as the qualitative promised then Mn would be expected to "leak" to the fiber orientation data in the figure are in close agreement. extracellular space and ultimately diffuse away from the The agreement between the Trace/3 values in the table enhancing region. Based on these considerations, the Mn- meant that the water diffusivities were similar in the labeling seen at the epicenter slide is consistent with the underlying tissues that these measurements were notion that some CST fibers might have maintained at obtained. The agreement between the FA values indicated least some level of connectivity across the injury. similar diffusion anisotropy, which indirectly suggested that the underlying tissues had axonal structure. Combin- To further support this interpretation, we performed dif- ing this with the observation that all the principle eigen- fusion tensor imaging (DTI) on the same slice locations. vectors were oriented along the cord provided alternative In previous studies of the rat optic tract, MEI and DTI have evidence that the Mn-labeled tissue at the epicenter was been used as complementary methods to confirm connec- part of the CST. The quantitative data in each column of tivity [14]. Accordingly, we expect that if the Mn-labeling the table can be seen as all comparing well. This agree- at the epicenter is truly associated with the underlying ment clearly provides alternative evidence that the Mn- connected fibers in the CST, then the water diffusion labeled tissue at the epicenter was part of the CST. The fib- Cigar Figure 5 -shaped ellipsoidal representation of the principal eigenvectors estimated from the DTI measurements Cigar-shaped ellipsoidal representation of the principal eigenvectors estimated from the DTI measurements. The eigenvector estimates from the Mn-labeled regions were only plotted on backgrounds that are the same as those rostral, epicenter and caudal images in Fig. 4. Therefore, the density of the vectors is associated with the size of the Mn-enhancement. Vector direc- tions are all aligned along the cord in all the three images. The direction of the alignment is consistent with the anatomical ori- entation of the descending neuronal fibers in the CST. The DTI data acquisition included first the baseline image, followed by the diffusion-weighted images obtained with applying diffusion sensitizing gradients along the directions (110,101,011,-110,- 101,0-11). Diffusion weighting was achieved using gradient strength = 80 mT/m, width (δ) = 6.5 ms and separation (Δ) = 11 ms 2 2 to produce b-value of b = 342 s/mm . Other parameters were TR/TE = 2500/26 ms, FOV = 10 × 10 mm , acquisition matrix = 128 × 128, slice thickness = 2 mm and NEX = 2. Page 6 of 8 (page number not for citation purposes) BMC Medical Imaging 2006, 6:15 http://www.biomedcentral.com/1471-2342/6/15 ers in CST are orientated parallel along the cord. These Competing interests demonstrations are important because they validate our The author(s) declare that they have no competing inter- DTI measurement protocols. This agreement is very ests. encouraging since it demonstrates the ability of two differ- ent modalities (functional MEI and structural DTI) to sen- Authors' contributions sitively detect the intact fibers in the injured SC. M.B. is responsible for the conception and design of the experiments as well as the analysis and interpretation of The use of IR imaging produces tissue contrast variations the data. dependent on the inversion time (TI). In IR acquisitions, the slight differences between the longitudinal relaxation Acknowledgements This work was supported by NIH Grants NS052610 and NS054019. The times (T1) in the GM and the WM (including the CST) can author thanks Dr. Yong-Yue He for his help in animal preparation and sur- be utilized to visualize the GM tissue as hypointense com- geries, Dr. Baraa Al-Hafez for his help in MRI scans, Rebecca Chambers for pared to the WM and CST, unlike the image contrast typi- her help in producing Fig. 1 and Josh Powell for his editorial help. The cally seen in the SE images of the SC in Fig. 3[10]. The author also greatly appreciates Dr. William M. Brooks for critically reading accumulation of Mn lowers the T1 in CST, introducing a the manuscript and providing his feedback. new contrast behavior seen in the IR image of the SC. We determined that our acquisition parameters provided a References good balance between image contrast (enhanced CST ver- 1. Dimitrijevic MR, Persy I, Forstner C, Kern H, Dimitrijevic MM: Motor control in the human spinal cord. Artif Organs 2005, sus GM and WM signal suppression) and acquisition time 29(3):216-219. at 9.4 T. Previous in vivo IR studies of the songbird brain 2. Bareyre FM, Kerschensteiner M, Raineteau O, Mettenleiter TC, at 7 T employed a somewhat different parameter set (T / Weinmann O, Schwab ME: The injured spinal cord spontane- ously forms a new intraspinal circuit in adult rats. Nat Neurosci T /T = 4000/20/855 ms) [11]. However, we note the var- E I 2004, 7(3):269-277. iations in the species and organs studied, the experimental 3. Raineteau O, Fouad K, Bareyre FM, Schwab ME: Reorganization of descending motor tracts in the rat spinal cord. Eur J Neurosci protocols followed and the magnetic field strength, each 2002, 16(9):1761-1771. of which contributes to the differences in imaging param- 4. Tsai EC, van Bendegem RL, Hwang SW, Tator CH: A novel method eters. for simultaneous anterograde and retrograde labeling of spi- nal cord motor tracts in the same animal. J Histochem Cytochem 2001, 49(9):1111-1122. It is also important to note that we have obtained high res- 5. Vercelli A, Repici M, Garbossa D, Grimaldi A: Recent techniques olution images using an inductively-coupled surface coil. for tracing pathways in the central nervous system of devel- oping and adult mammals. Brain Res Bull 2000, 51(1):11-28. This coil inherently produces an inhomogeneous rf field 6. Bareyre FM, Kerschensteiner M, Misgeld T, Sanes JR: Transgenic which ultimately yields spatially variant inversion. We labeling of the corticospinal tract for monitoring axonal responses to spinal cord injury. Nat Med 2005, notice that this may be an issue in the IR-MEI acquisition 11(12):1355-1360. and lead to an incomplete suppression of the background 7. Bilgen M, Dancause N, Al-Hafez B, He YH, Malone TM: Manganese- tissue during the inversion recovery unless the power of enhanced MRI of Rat Spinal Cord injury. Magn Reson Imaging In press 2005. the 90° rf pulse is adjusted carefully to focus on the SC. 8. Bilgen M, Peng W, Al-Hafez B, Dancause N, He YY, Cheney PD: Elec- trical stimulation of cortex improves corticospinal tract tracing in rat spinal cord using manganese-enhanced MRI. J Conclusion Neurosci Methods 2006. This paper outlines the procedures required for success- 9. Bilgen M: A new device for experimental modeling of central fully tracing the CST using MEI and demonstrates the nervous system injuries. Neurorehabil Neural Repair 2005, 19(3):219-226. potential of MEI in indirectly assessing the axonal fiber 10. Bilgen M, Al-Hafez B, T MM, I VS: Ex vivo magnetic resonance connectivity in injured rat SC. Further work is, however, imaging of rat spinal cord at 9.4 T. Magn Reson Imaging 2005, warranted to confirm the reliability of tract tracing based 23(4):601-605. 11. Tindemans I, Boumans T, Verhoye M, Van der Linden A: IR-SE and on MEI using gold-standard histological analysis such as IR-MEMRI allow in vivo visualization of oscine neuroarchitec- using transgenic [6] or anterograde tracer labeling ture including the main forebrain regions of the song control system. NMR Biomed 2006, 19(1):18-29. approaches [7]. Once the veracity of the methods is con- 12. Bilgen M: Simple, low-cost multipurpose RF coil for MR firmed, future studies aiming to address different issues microscopy at 9.4 T. Magn Reson Med 2004, 52(4):937-940. such as the ability to examine the CST connectivity in 13. Paxinos G: The Rat Nervous System, Academic Press, Lon- don. 1995. more severe injuries or to explore the role of dynamic 14. Lin CP, Tseng WY, Cheng HC, Chen JH: Validation of diffusion imaging of intensity build-up in the CST below the injury tensor magnetic resonance axonal fiber imaging with regis- tered manganese-enhanced optic tracts. Neuroimage 2001, with time will be possible. The data from such studies may 14(5):1035-1047. be used to assess the density and distribution of the con- 15. Bilgen M, Narayana PA: Mohr diagram interpretation of aniso- tinuous fibers or to evaluate the efficacy of promoting the tropic diffusion indices in MRI. Magn Reson Imaging 2003, 21(5):567-572. fiber connectivity in injured SC with endogeneous or exo- geneous interventions. 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Imaging corticospinal tract connectivity in injured rat spinal cord using manganese-enhanced MRI

BMC Medical Imaging , Volume 6 (1) – Nov 17, 2006

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Springer Journals
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Copyright © 2006 by Bilgen; licensee BioMed Central Ltd.
Subject
Medicine & Public Health; Imaging / Radiology
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1471-2342
DOI
10.1186/1471-2342-6-15
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17112375
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Abstract

Background: Manganese-enhanced MRI (MEI) offers a novel neuroimaging modality to trace corticospinal tract (CST) in live animals. This paper expands this capability further and tests the utility of MEI to image axonal fiber connectivity in CST of injured spinal cord (SC). Methods: A rat was injured at the thoracic T4 level of the SC. The CST was labeled with manganese (Mn) injected intracortically at two weeks post injury. Next day, the injured SC was imaged using MEI and diffusion tensor imaging (DTI) modalities. Results: In vivo MEI data obtained from cervical SC confirmed that CST was successfully labeled with Mn. Ex vivo MEI data obtained from excised SC depicted Mn labeling of the CST in SC sections caudal to the lesion, which meant that Mn was transported through the injury, possibly mediated by viable CST fibers present at the injury site. Examining the ex vivo data from the injury epicenter closely revealed a thin strip of signal enhancement located ventrally between the dorsal horns. This enhancement was presumably associated with the Mn accumulation in these intact fibers projecting caudally as part of the CST. Additional measurements with DTI supported this view. Conclusion: Combining these preliminary results collectively demonstrated the feasibility of imaging fiber connectivity in experimentally injured SC using MEI. This approach may play important role in future investigations aimed at understanding the neuroplasticity in experimental SCI research. motor neurons in the SC [1]. Because, many different Background Spinal cord injury (SCI) disrupts the functional integrity types of movements are controlled through the fibers of of neuronal circuits in the ascending and descending the CST, understanding how this tract adapts to SCI is the pathways in spinal cord (SC), thereby compromising the focus of current experimental efforts [2,3]. A number of voluntary motor control of muscles below the site of the methods have been developed to anatomically trace the injury and the conduction of sensory signals from distal CST fibers using different neuronal tracers and tract trac- inputs. In SCI research, a particular pathway of interest is ing procedures [4,5]. However, these conventional meth- the corticospinal tract (CST) as it is the major long ods require elaborate tissue analysis based on histology descending output connecting the cerebral cortex with the and immunohistochemistry, and therefore preclude Page 1 of 8 (page number not for citation purposes) BMC Medical Imaging 2006, 6:15 http://www.biomedcentral.com/1471-2342/6/15 repeated measurements from the same animal. One strat- mal to confirm that the CST labeling in the SC was suc- egy to reduce the number of post mortem procedure is the cessful, and on excised cords to obtain high resolution use of a transgenic animal model that has recently been MEI data around the lesion. Here, we briefly summarize engineered to specifically and completely label the CST these procedures and give the details of the individual fibers by yellow fluorescent protein [6]. Although, this components critical to the current work. novel development simplifies the work and accelerates Spinal Cord Injury (SCI) the assessment of the regeneration and remodeling of the neuronal connections in the injured SC, it still requires Image artifacts from heart motion obscure the 3D visuali- invasive post mortem examination, making it unfit for zation of the SCI at levels below the thoracic T4 level dynamic imaging of live preparations. when the signal is acquired using volume coil. Therefore, SCI was induced at the T4 level to minimize these artifacts As an alternative method, we have shown that manga- by following the injury protocol described elsewhere [9]. nese-enhanced magnetic resonance imaging (MEI) can Briefly, the rat was anesthetized using spontaneous inha- offer new possibilities in probing the SC in live animals lation of 4% isoflurane for induction and maintained on [7,8]. MEI is an experimental technique that uses the par- a mixture of 1.5% isoflurane, 30% oxygen, and air deliv- 2+ amagnetic manganese ion (Mn ) as a contrast agent. ered through a nose mask. A rectangular area was shaved With an ionic radius similar to that of calcium-ion, Mn on the back and an incision was made to expose the pos- can enter cells through the voltage-gated calcium chan- terior elements of the spine. Then, a rongeur was used to nels, and then remain confined to the intracellular com- perform a laminectomy at T4 to expose the spinal cord, partment of intact axons. After administering Mn to the but to leave the dura intact. After stabilizing the SC with biological system, uptake and transport in the tissue of two forceps attached to the rostral T3 and caudal T5 verte- interest can be monitored remotely using in vivo magnetic bral bodies, the laminectomized section was positioned resonance imaging protocols sensitive to Mn-induced under the impactor tip of the injury device [9]. Contusion altered relaxation characteristics. Applying Mn directly injury was produced by using a rectangular (1 mm × 2 into the SC just rostral to a hemisectioned SC, we have mm) injury tip with velocity of 1.5 m/s and contact dura- demonstrated that MEI can potentially play an important tion of 80 ms. The deformation depth was set to 0.5 mm role in mapping viable neuronal tissue at and below the for producing mild partial injury with good prognosis for site of injury because functional tissue on the uninjured behavioral improvement. Next, the skin was closed and side takes up Mn and hence is delineated on the MEI [7]. the animal was placed in a heated cage to maintain the Moreover, after intracortical administration of Mn into body temperature while recovering. the motor areas of the brain and stimulating the cortex electrically, we have produced robust and detectable Intracortical Mn delivery anterograde Mn labeling of the CST from cortex caudally Intracortical delivery of Mn was achieved as described in to the thoracic SC levels [8]. The aim of the current study [8]. On post injury day 14, the injured rat was anesthe- is determine whether MEI can detect CST connectivity in tized again using ketamine hydrochloride delivered intra- partially injured SC. We used a rat animal model of mild muscularly at an initial dose of 150 mg/kg, followed by thoracic SCI. This model produces an incomplete injury additional injections at doses of 5–20 mg/kg, as needed. with functionally intact CST fibers remaining at the injury The head of the anesthetized rat was fixed in a stereotaxic site [9]. We now demonstrate that these fibers provide an frame (Kopf Instruments, Tujunga, CA). A midline inci- environment for transporting Mn caudal to the lesion. sion was made on the scalp from approximately 2.5 mm Visualization of the Mn-labeled tract below the lesion rostral to 7.5 mm caudal to the bregma, and the skin was using MEI therefore provides a method to detect the fibers retracted with hemostats. Bilateral 1.0 mm diameter burr projecting through the injured section and hence fiber holes were drilled into the skull 1.5 mm rostral to the connectivity in injured SC. bregma and 2.0 mm lateral to the midline using a 1 mm diameter trephine bit attached to a dental drill. An addi- Methods tional craniotomy was performed on one side of the skull The experiments were conducted on one Sprague-Dawley at a location 6.5 mm caudal to the bregma and 2.0 mm rat (~300 g) under a protocol approved by the University lateral to the midline. Through this opening, a 2.0 mm of Kansas Medical Center Institutional Animal Care and diameter titanium screw was inserted until it rested on the Use Committee. We followed established procedures dura. This screw served as the reference electrode for the described previously for the surgery, SCI, Mn delivery and electrical stimulation. MEI scans [8-10]. After SCI, the rat was left to recover for two weeks. On post injury day 14, Mn was delivered intra- Using 1 M MnCl in a 1 μL Hamilton syringe with a cortically and the motor cortex was stimulated electrically. tapered, graduated micropipette tip, a direct stereotaxical The next day, MEI scans were performed on the living ani- injection was made to deliver a total of 0.2 μL of this solu- Page 2 of 8 (page number not for citation purposes) BMC Medical Imaging 2006, 6:15 http://www.biomedcentral.com/1471-2342/6/15 tion focally at 0.5 mm below the surface of the cortex bilities in visualizing the Mn-labeled CST in addition to through the center of the first burr hole. The solution was the 3D GE imaging. injected slowly over a period of five minutes. To prevent backflow, the pipette was left in place for another five The MRI scans were performed 24 hr after the Mn-delivery minutes prior to withdrawal. This procedure was repeated and electrical stimulation. The rat was anesthetized using for the contra lateral motor area through the second burr spontaneous inhalation of 4% isoflurane for induction, hole. followed by a mixture of 1.5% isoflurane, 30% oxygen, and air delivered through a nose mask. The head was sta- Cortical stimulation bilized on a Plexiglas holder and positioned in a 6 cm Following the Mn delivery, electrical stimulation of the inner diameter volume coil for MRI scanning on a 9.4 T cortex was achieved with a 1 mm diameter stainless steel horizontal Varian scanner (Varian Inc., Palo Alto, CA). electrode and the titanium screw serving as the reference. While in the scanner, the physiological condition of the The electrode was connected to a DS7 Digitimer constant rat was monitored using ECG, respiratory and tempera- current stimulator (Digitimer Ltd., Hertfordshire, Eng- ture probes connected to a MR-compatible small animal land) and lowered through the first burr hole on the same monitoring and gating system (Model 1025, SA Instru- side as the screw until it touched to dura. A Grass S48 ments, Inc., Stony Brook, NY). The body temperature was stimulator (Grass Medical Instruments, Quincy, MA) gen- maintained at 37°C by circulating warm air with 40 % erated the stimulus pulse sequence used to trigger the con- humidity using a 5 cm diameter plastic tube fitted to the stant current stimulator. Trains of biphasic stimuli (each back door of the magnet bore. phase 0.2 ms, negative first) were applied at 100 Hz for an "on" period of 5 seconds followed by an "off" period of 5 After confirming the placement of the animal in the mag- seconds. The current was adjusted until an evoked visible net's isocenter with scout images, T -weighted volumetric motor response was produced in the forelimb, hindlimb images covering the brain and SC at the cervical and tho- or tail, and applied for 90 minutes. If the strength of the racic levels were acquired using a 3D GE sequence (T /T R E evoked movement declined noticeably, frequency and = 45/4 ms and flip angle (FA) = 45°. The data were sam- current values were increased to maintain a constant pled on a matrix = 128 × 128 × 64 ranging over a volume , and processed and interpolated to motor response. These procedures were then repeated for of 70 × 32 × 32 mm the opposite cortex through a second burr hole. Immedi- 256 × 256 × 128 pixels for the final display. Maximum ately after stimulation, the electrodes were removed, and intensity projections were generated to delineate the Mn the skin was sutured tightly. The animal was then left to enhancement in the CST over a desired thickness in the recover in its cage. sagittal plane. Then, a sagittal IR-MEI was acquired (T /T / R E T = 2000/12/550 ms, field-of-view (FOV) = 70 × 32 mm , Magnetic resonance imaging image matrix = 256 × 128, slice thickness = 2 mm and The paramagnetic nature of Mn changes the MR proper- NEX = 4). Finally, axial IR-MEI was acquired (T /T /T = R E I ties of the tissue where it is accumulated. In particular, sig- 2000/17/550 ms, field-of-view (FOV) = 22 × 22 mm , nals from these regions are enhanced on conventional T1- image matrix = 128 × 128, slice thickness = 2 mm and weighted spin echo (SE) images. The CST in rat SC is ana- NEX = 4). The rat was then removed from the scanner, tomically located in the ventral-most part of the dorsal euthanized using cardiac puncture and the vertebral body funiculus of the SC, i.e., near the central canal between the was dissected from the animal. The excised sample with dorsal horns of the gray matter (GM). Because of this top- intact spine was scanned ex vivo at room temperature ological arrangement, Mn-labeled CST becomes difficult using an inductively coupled surface coil centered at the to differentiate from the GM on the SE image since both injury epicenter [10,12]. High resolution multi-slice SE structures exhibit similar intensity [8]. The use of the 3D images were acquired in sagittal and axial planes (sagittal gradient-echo (GE) sequence with short repetition time parameters: T /T = 2500/12 ms, field-of-view = 32 × 10 R E however overcomes this limitation and produces robust mm , image matrix = 256 × 128, slice thickness = 0.5 mm and detectible Mn-labeled CST in SC. More recently, and NEX = 2; axial parameters: T /T = 2500/12 ms, field- R E inversion recovery SE (IR-SE) acquisitions were shown to of-view = 10 × 10 mm , image matrix = 128 × 128, slice offer better sensitivity to Mn in neuronal tissue [11]. Pre- thickness = 2 mm and NEX = 2). Finally, axial IR-MEI of viously, we have used IR-SE imaging to demonstrate the excised spine and SC were acquired (T /T /T = 2000/ R E I quantitatively that the T1-relaxation times of the GM and 15/550 ms, field-of-view (FOV) = 10 × 10 mm , image white matter (WM) are indeed slightly different in rat SC matrix = 128 × 128, slice thickness = 2 mm and NEX = 4). [10]. Based on the promise that IR-SE provides richer con- trast enhancement and our experience with this sequence, Results and discussion we also performed IR-SE imaging to demonstrate its capa- The CST in rat runs caudally from cortex through internal capsule, cerebral peduncle, longitudinal pontine fascicu- Page 3 of 8 (page number not for citation purposes) BMC Medical Imaging 2006, 6:15 http://www.biomedcentral.com/1471-2342/6/15 labeled CST centered within the SC and lesion as deline- ated by a single patch of signal hypointensity. Figure 2b shows the transverse section of the cervical SC from the imaging plane marked with a thin blue rectangle on the sagittal image. This image shows a nearly circular region of signal hyperintensity located centrally within the SC. This bright region with a well-defined boundary repre- sents the CST labeled with Mn. The corresponding data in Figs. 2c and 2d were acquired with IR-MEI and show the capability of this approach to produce in vivo signal con- trast delineating the CST. The lesion was however difficult to visualize on the IR-MEI in sagittal view. Nevertheless, Th system and projection of way Figure 1 ree-dimensional visualiz the atiocorticospinal tract n of the rat central n (CST) path- ervous Three-dimensional visualization of the rat central nervous these volumetric data confirmed that the above experi- system and projection of the corticospinal tract (CST) path- mental procedures were successful in terms of labeling the way. The data were acquired using 3D GE-MEI after 24 h of CST with Mn in live animal. intracortical injection of Mn and electrical stimulation of the motor cortex. The red color denotes the Mn-enhancement, After this confirmation, the experiment was continued labeling the CST in brain and spinal cord. with scans on the excised cord to get high resolution data. The resulting ex vivo images from this effort are shown in Fig. 3. The sagittal views in Figs. 3a and 3b show the injury lus, pyramid, pyramidal decussation, and descends in the and its extent along the SC. The anatomical images from dorsal fasciculus of the SC [13]. Figure 1 visualizes the the selected axial planes (blue rectangles in Fig. 3b) shown spatial projection of this tract in relation to the overall in Figs. 3c,d, and 3e depict the injured tissue morphology central nervous system by volume rendering of the in greater detail at the epicenter as well as the normal cord acquired GE-MEI data in 3D. The Mn-enhancement in the at sections rostral and caudal to the injury site. Figure 4 figure is represented in red. Figure 2 depicts the Mn- shows the IR-MEIs acquired from the same axial slice posi- enhancement in 2D views. The image in Fig. 2a shows a tions. The GM on these images appears relatively darker single sagittal slice depicting the Mn injection site, Mn- compared to the WM in normal sections of the cord. In In vivo pl Figure 2 anes visualization of the rat central nervous system and cross sectional views of the CST pathway in the sagittal and axial In vivo visualization of the rat central nervous system and cross sectional views of the CST pathway in the sagittal and axial planes. This data were acquired using 3D GE-MEI (a and b) and IR-MEI (c and d). The arrow labeled "SCI" points to the lesion at the T4 level. Thin-rectangles overlaid on the sagittal images represent the slice orientation for the axial images. Arrowhead in a denotes the site of the Mn injection in the brain, where the signal hypointensity is due to the presence of high local concen- tration of Mn. SI – primary somatosensory cortex, ic – internal capsule, thal – thalamus, cp – cerebral peduncle and py – pyramidal tract. Page 4 of 8 (page number not for citation purposes) BMC Medical Imaging 2006, 6:15 http://www.biomedcentral.com/1471-2342/6/15 a (right) Figure 3 , b) Ex vivo spin echo ima direction in sagittal planes ges showing the injury (arrow head) in an excised SC but intact spine from rostral (left) to caudal a, b) Ex vivo spin echo images showing the injury (arrow head) in an excised SC but intact spine from rostral (left) to caudal (right) direction in sagittal planes. c, d, e) Ex vivo anatomical axial spin echo images of the spinal cord from the three slice loca- tions (c – rostral to injury, d – injury epicenter and e – caudal to injury) depicted by the thin-rectangles overlaid on the sagittal image in b. Fig. 4a, the pattern of signal enhancement rostral to the situated right posterior to the central canal between the injury can be seen as confined spatially to the ventral- dorsal roots. Signal enhancement is also seen in Fig. 4c at most part of the dorsal funiculus between the dorsal the expected CST location caudal to the injury. Interpreta- horns of the GM in sections rostral and caudal the injury. tion of these results requires consideration of possible This enhanced region overlaps exactly with the expected mechanisms that might facilitate trans-injury propagation anatomical location of the CST in rat SC [8,13]. Careful of the Mn-dependent contrast. Likely mechanisms include examination of the image from the injury epicenter (Fig. axonal transport and extracellular diffusion of Mn. If the 4b) reveals a thin strip of signal enhancement, which is transport were by purely extracellular diffusion, we would E Figure 4 x vivo IR-MEIs from the same slice orientations as those images in Figs. 3-c, d and e Ex vivo IR-MEIs from the same slice orientations as those images in Figs. 3-c, d and e. a) Mn-enhanced CST rostral to the injury. b) Mn-enhancement at the epicenter of the injury. c) Mn-labelling of the CST caudal to the injury. The partial signal enhance- ment, depicted by the arrowhead in b, is likely to represent a portion of the CST that is populated with intact fibers. By contin- uously projecting through the injury site, these intact fibers transport Mn from rostral to caudal sections, as indicated by the presence of focal signal enhancement in c. Page 5 of 8 (page number not for citation purposes) BMC Medical Imaging 2006, 6:15 http://www.biomedcentral.com/1471-2342/6/15 Table 1: Measurements from Mn-labeled regions, above and below the lesion, and at the epicenter of the injury. Above the lesion Epicenter Below the lesion -3 2 Trace/3 (× 10 mm /s) 0.69 ± 0.05 0.59 ± 0.07 0.64 ± 0.06 FA 0.88 ± 0.08 0.79 ± 0.13 0.85 ± 0.10 Trace/3: average of the three principal eigenvalues of the diffusion tensor. FA: fractional anisotropy index. The statistics are given as mean ± standard deviation. expect that the axial extent of the enhancement region properties measured within this region would match with would expand gradually from rostral to caudal, perhaps those obtained from above and below the lesion. Process- eventually including the complete cord. However, we ing the acquired DTI data, pixel-by-pixel, produced esti- observed enhancement only in the location expected to be mates for the mean water diffusivity, diffusion anisotropy occupied by the fibers leading from the CST to distal por- and diffusion direction as respectively represented by the tions of the cord, even in the images obtained caudal to Trace/3 (average of the diffusion tensor eigenvalues) and the injury. Since the Mn enhancement remains closely fractional anisotropy (FA) parameters and principal confined to the location of the fibers into which it was eigenvector [15]. The results from these computations are originally administered, we speculate that this indicates a given in Table 1 and in Fig. 5. The quantitative Trace/3 and high level of axonal integrity since if the axons are com- FA data in each row of the table as well as the qualitative promised then Mn would be expected to "leak" to the fiber orientation data in the figure are in close agreement. extracellular space and ultimately diffuse away from the The agreement between the Trace/3 values in the table enhancing region. Based on these considerations, the Mn- meant that the water diffusivities were similar in the labeling seen at the epicenter slide is consistent with the underlying tissues that these measurements were notion that some CST fibers might have maintained at obtained. The agreement between the FA values indicated least some level of connectivity across the injury. similar diffusion anisotropy, which indirectly suggested that the underlying tissues had axonal structure. Combin- To further support this interpretation, we performed dif- ing this with the observation that all the principle eigen- fusion tensor imaging (DTI) on the same slice locations. vectors were oriented along the cord provided alternative In previous studies of the rat optic tract, MEI and DTI have evidence that the Mn-labeled tissue at the epicenter was been used as complementary methods to confirm connec- part of the CST. The quantitative data in each column of tivity [14]. Accordingly, we expect that if the Mn-labeling the table can be seen as all comparing well. This agree- at the epicenter is truly associated with the underlying ment clearly provides alternative evidence that the Mn- connected fibers in the CST, then the water diffusion labeled tissue at the epicenter was part of the CST. The fib- Cigar Figure 5 -shaped ellipsoidal representation of the principal eigenvectors estimated from the DTI measurements Cigar-shaped ellipsoidal representation of the principal eigenvectors estimated from the DTI measurements. The eigenvector estimates from the Mn-labeled regions were only plotted on backgrounds that are the same as those rostral, epicenter and caudal images in Fig. 4. Therefore, the density of the vectors is associated with the size of the Mn-enhancement. Vector direc- tions are all aligned along the cord in all the three images. The direction of the alignment is consistent with the anatomical ori- entation of the descending neuronal fibers in the CST. The DTI data acquisition included first the baseline image, followed by the diffusion-weighted images obtained with applying diffusion sensitizing gradients along the directions (110,101,011,-110,- 101,0-11). Diffusion weighting was achieved using gradient strength = 80 mT/m, width (δ) = 6.5 ms and separation (Δ) = 11 ms 2 2 to produce b-value of b = 342 s/mm . Other parameters were TR/TE = 2500/26 ms, FOV = 10 × 10 mm , acquisition matrix = 128 × 128, slice thickness = 2 mm and NEX = 2. Page 6 of 8 (page number not for citation purposes) BMC Medical Imaging 2006, 6:15 http://www.biomedcentral.com/1471-2342/6/15 ers in CST are orientated parallel along the cord. These Competing interests demonstrations are important because they validate our The author(s) declare that they have no competing inter- DTI measurement protocols. This agreement is very ests. encouraging since it demonstrates the ability of two differ- ent modalities (functional MEI and structural DTI) to sen- Authors' contributions sitively detect the intact fibers in the injured SC. M.B. is responsible for the conception and design of the experiments as well as the analysis and interpretation of The use of IR imaging produces tissue contrast variations the data. dependent on the inversion time (TI). In IR acquisitions, the slight differences between the longitudinal relaxation Acknowledgements This work was supported by NIH Grants NS052610 and NS054019. The times (T1) in the GM and the WM (including the CST) can author thanks Dr. Yong-Yue He for his help in animal preparation and sur- be utilized to visualize the GM tissue as hypointense com- geries, Dr. Baraa Al-Hafez for his help in MRI scans, Rebecca Chambers for pared to the WM and CST, unlike the image contrast typi- her help in producing Fig. 1 and Josh Powell for his editorial help. The cally seen in the SE images of the SC in Fig. 3[10]. The author also greatly appreciates Dr. William M. Brooks for critically reading accumulation of Mn lowers the T1 in CST, introducing a the manuscript and providing his feedback. new contrast behavior seen in the IR image of the SC. We determined that our acquisition parameters provided a References good balance between image contrast (enhanced CST ver- 1. Dimitrijevic MR, Persy I, Forstner C, Kern H, Dimitrijevic MM: Motor control in the human spinal cord. Artif Organs 2005, sus GM and WM signal suppression) and acquisition time 29(3):216-219. at 9.4 T. Previous in vivo IR studies of the songbird brain 2. Bareyre FM, Kerschensteiner M, Raineteau O, Mettenleiter TC, at 7 T employed a somewhat different parameter set (T / Weinmann O, Schwab ME: The injured spinal cord spontane- ously forms a new intraspinal circuit in adult rats. Nat Neurosci T /T = 4000/20/855 ms) [11]. However, we note the var- E I 2004, 7(3):269-277. iations in the species and organs studied, the experimental 3. Raineteau O, Fouad K, Bareyre FM, Schwab ME: Reorganization of descending motor tracts in the rat spinal cord. Eur J Neurosci protocols followed and the magnetic field strength, each 2002, 16(9):1761-1771. of which contributes to the differences in imaging param- 4. Tsai EC, van Bendegem RL, Hwang SW, Tator CH: A novel method eters. for simultaneous anterograde and retrograde labeling of spi- nal cord motor tracts in the same animal. J Histochem Cytochem 2001, 49(9):1111-1122. It is also important to note that we have obtained high res- 5. Vercelli A, Repici M, Garbossa D, Grimaldi A: Recent techniques olution images using an inductively-coupled surface coil. for tracing pathways in the central nervous system of devel- oping and adult mammals. Brain Res Bull 2000, 51(1):11-28. This coil inherently produces an inhomogeneous rf field 6. Bareyre FM, Kerschensteiner M, Misgeld T, Sanes JR: Transgenic which ultimately yields spatially variant inversion. We labeling of the corticospinal tract for monitoring axonal responses to spinal cord injury. Nat Med 2005, notice that this may be an issue in the IR-MEI acquisition 11(12):1355-1360. and lead to an incomplete suppression of the background 7. Bilgen M, Dancause N, Al-Hafez B, He YH, Malone TM: Manganese- tissue during the inversion recovery unless the power of enhanced MRI of Rat Spinal Cord injury. Magn Reson Imaging In press 2005. the 90° rf pulse is adjusted carefully to focus on the SC. 8. Bilgen M, Peng W, Al-Hafez B, Dancause N, He YY, Cheney PD: Elec- trical stimulation of cortex improves corticospinal tract tracing in rat spinal cord using manganese-enhanced MRI. J Conclusion Neurosci Methods 2006. This paper outlines the procedures required for success- 9. Bilgen M: A new device for experimental modeling of central fully tracing the CST using MEI and demonstrates the nervous system injuries. Neurorehabil Neural Repair 2005, 19(3):219-226. potential of MEI in indirectly assessing the axonal fiber 10. Bilgen M, Al-Hafez B, T MM, I VS: Ex vivo magnetic resonance connectivity in injured rat SC. Further work is, however, imaging of rat spinal cord at 9.4 T. Magn Reson Imaging 2005, warranted to confirm the reliability of tract tracing based 23(4):601-605. 11. Tindemans I, Boumans T, Verhoye M, Van der Linden A: IR-SE and on MEI using gold-standard histological analysis such as IR-MEMRI allow in vivo visualization of oscine neuroarchitec- using transgenic [6] or anterograde tracer labeling ture including the main forebrain regions of the song control system. NMR Biomed 2006, 19(1):18-29. approaches [7]. Once the veracity of the methods is con- 12. Bilgen M: Simple, low-cost multipurpose RF coil for MR firmed, future studies aiming to address different issues microscopy at 9.4 T. Magn Reson Med 2004, 52(4):937-940. such as the ability to examine the CST connectivity in 13. Paxinos G: The Rat Nervous System, Academic Press, Lon- don. 1995. more severe injuries or to explore the role of dynamic 14. Lin CP, Tseng WY, Cheng HC, Chen JH: Validation of diffusion imaging of intensity build-up in the CST below the injury tensor magnetic resonance axonal fiber imaging with regis- tered manganese-enhanced optic tracts. Neuroimage 2001, with time will be possible. The data from such studies may 14(5):1035-1047. be used to assess the density and distribution of the con- 15. Bilgen M, Narayana PA: Mohr diagram interpretation of aniso- tinuous fibers or to evaluate the efficacy of promoting the tropic diffusion indices in MRI. Magn Reson Imaging 2003, 21(5):567-572. fiber connectivity in injured SC with endogeneous or exo- geneous interventions. Page 7 of 8 (page number not for citation purposes) BMC Medical Imaging 2006, 6:15 http://www.biomedcentral.com/1471-2342/6/15 Pre-publication history The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-2342/6/15/prepub Publish with Bio Med Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical researc h in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright BioMedcentral Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp Page 8 of 8 (page number not for citation purposes)

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