Imaging corticospinal tract connectivity in injured rat spinal cord using manganese-enhanced MRI
© Bilgen; licensee BioMed Central Ltd. 2006
Received: 29 May 2006
Accepted: 17 November 2006
Published: 17 November 2006
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).
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.
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.
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.
Spinal cord injury (SCI) disrupts the functional integrity of neuronal circuits in the ascending and descending pathways in spinal cord (SC), thereby compromising the voluntary motor control of muscles below the site of the injury and the conduction of sensory signals from distal inputs. In SCI research, a particular pathway of interest is the corticospinal tract (CST) as it is the major long descending output connecting the cerebral cortex with the motor neurons in the SC . Because, many different types of movements are controlled through the fibers of the CST, understanding how this tract adapts to SCI is the focus of current experimental efforts [2, 3]. A number of methods have been developed to anatomically trace the CST fibers using different neuronal tracers and tract tracing procedures [4, 5]. However, these conventional methods require elaborate tissue analysis based on histology and immunohistochemistry, and therefore preclude repeated measurements from the same animal. One strategy to reduce the number of post mortem procedure is the use of a transgenic animal model that has recently been engineered to specifically and completely label the CST fibers by yellow fluorescent protein . Although, this novel development simplifies the work and accelerates the assessment of the regeneration and remodeling of the neuronal connections in the injured SC, it still requires invasive post mortem examination, making it unfit for dynamic imaging of live preparations.
As an alternative method, we have shown that manganese-enhanced magnetic resonance imaging (MEI) can offer new possibilities in probing the SC in live animals [7, 8]. MEI is an experimental technique that uses the paramagnetic manganese ion (Mn2+) as a contrast agent. With an ionic radius similar to that of calcium-ion, Mn can enter cells through the voltage-gated calcium channels, and then remain confined to the intracellular compartment of intact axons. After administering Mn to the biological system, uptake and transport in the tissue of interest can be monitored remotely using in vivo magnetic resonance imaging protocols sensitive to Mn-induced altered relaxation characteristics. Applying Mn directly into the SC just rostral to a hemisectioned SC, we have demonstrated that MEI can potentially play an important role in mapping viable neuronal tissue at and below the site of injury because functional tissue on the uninjured side takes up Mn and hence is delineated on the MEI . Moreover, after intracortical administration of Mn into the motor areas of the brain and stimulating the cortex electrically, we have produced robust and detectable anterograde Mn labeling of the CST from cortex caudally to the thoracic SC levels . The aim of the current study is determine whether MEI can detect CST connectivity in partially injured SC. We used a rat animal model of mild thoracic SCI. This model produces an incomplete injury with functionally intact CST fibers remaining at the injury site . We now demonstrate that these fibers provide an environment for transporting Mn caudal to the lesion. Visualization of the Mn-labeled tract below the lesion using MEI therefore provides a method to detect the fibers projecting through the injured section and hence fiber connectivity in injured SC.
The experiments were conducted on one Sprague-Dawley rat (~300 g) under a protocol approved by the University of Kansas Medical Center Institutional Animal Care and Use Committee. We followed established procedures described previously for the surgery, SCI, Mn delivery and MEI scans [8–10]. After SCI, the rat was left to recover for two weeks. On post injury day 14, Mn was delivered intracortically and the motor cortex was stimulated electrically. The next day, MEI scans were performed on the living animal to confirm that the CST labeling in the SC was successful, and on excised cords to obtain high resolution MEI data around the lesion. Here, we briefly summarize these procedures and give the details of the individual components critical to the current work.
Spinal Cord Injury (SCI)
Image artifacts from heart motion obscure the 3D visualization of the SCI at levels below the thoracic T4 level when the signal is acquired using volume coil. Therefore, SCI was induced at the T4 level to minimize these artifacts by following the injury protocol described elsewhere . Briefly, the rat was anesthetized using spontaneous inhalation of 4% isoflurane for induction and maintained on a mixture of 1.5% isoflurane, 30% oxygen, and air delivered through a nose mask. A rectangular area was shaved on the back and an incision was made to expose the posterior elements of the spine. Then, a rongeur was used to perform a laminectomy at T4 to expose the spinal cord, but to leave the dura intact. After stabilizing the SC with two forceps attached to the rostral T3 and caudal T5 vertebral bodies, the laminectomized section was positioned under the impactor tip of the injury device . Contusion injury was produced by using a rectangular (1 mm × 2 mm) injury tip with velocity of 1.5 m/s and contact duration of 80 ms. The deformation depth was set to 0.5 mm for producing mild partial injury with good prognosis for behavioral improvement. Next, the skin was closed and the animal was placed in a heated cage to maintain the body temperature while recovering.
Intracortical Mn delivery
Intracortical delivery of Mn was achieved as described in . On post injury day 14, the injured rat was anesthetized again using ketamine hydrochloride delivered intramuscularly at an initial dose of 150 mg/kg, followed by additional injections at doses of 5–20 mg/kg, as needed. The head of the anesthetized rat was fixed in a stereotaxic frame (Kopf Instruments, Tujunga, CA). A midline incision was made on the scalp from approximately 2.5 mm rostral to 7.5 mm caudal to the bregma, and the skin was retracted with hemostats. Bilateral 1.0 mm diameter burr holes were drilled into the skull 1.5 mm rostral to the bregma and 2.0 mm lateral to the midline using a 1 mm diameter trephine bit attached to a dental drill. An additional craniotomy was performed on one side of the skull at a location 6.5 mm caudal to the bregma and 2.0 mm lateral to the midline. Through this opening, a 2.0 mm diameter titanium screw was inserted until it rested on the dura. This screw served as the reference electrode for the electrical stimulation.
Using 1 M MnCl2 in a 1 μL Hamilton syringe with a tapered, graduated micropipette tip, a direct stereotaxical injection was made to deliver a total of 0.2 μL of this solution focally at 0.5 mm below the surface of the cortex through the center of the first burr hole. The solution was injected slowly over a period of five minutes. To prevent backflow, the pipette was left in place for another five minutes prior to withdrawal. This procedure was repeated for the contra lateral motor area through the second burr hole.
Following the Mn delivery, electrical stimulation of the cortex was achieved with a 1 mm diameter stainless steel electrode and the titanium screw serving as the reference. The electrode was connected to a DS7 Digitimer constant current stimulator (Digitimer Ltd., Hertfordshire, England) and lowered through the first burr hole on the same side as the screw until it touched to dura. A Grass S48 stimulator (Grass Medical Instruments, Quincy, MA) generated the stimulus pulse sequence used to trigger the constant current stimulator. Trains of biphasic stimuli (each 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 seconds. The current was adjusted until an evoked visible motor response was produced in the forelimb, hindlimb or tail, and applied for 90 minutes. If the strength of the evoked movement declined noticeably, frequency and current values were increased to maintain a constant motor response. These procedures were then repeated for the opposite cortex through a second burr hole. Immediately after stimulation, the electrodes were removed, and the skin was sutured tightly. The animal was then left to recover in its cage.
Magnetic resonance imaging
The paramagnetic nature of Mn changes the MR properties of the tissue where it is accumulated. In particular, signals from these regions are enhanced on conventional T1-weighted spin echo (SE) images. The CST in rat SC is anatomically located in the ventral-most part of the dorsal funiculus of the SC, i.e., near the central canal between the dorsal horns of the gray matter (GM). Because of this topological arrangement, Mn-labeled CST becomes difficult to differentiate from the GM on the SE image since both structures exhibit similar intensity . The use of the 3D gradient-echo (GE) sequence with short repetition time however overcomes this limitation and produces robust and detectible Mn-labeled CST in SC. More recently, inversion recovery SE (IR-SE) acquisitions were shown to offer better sensitivity to Mn in neuronal tissue . Previously, we have used IR-SE imaging to demonstrate quantitatively that the T1-relaxation times of the GM and white matter (WM) are indeed slightly different in rat SC . Based on the promise that IR-SE provides richer contrast enhancement and our experience with this sequence, we also performed IR-SE imaging to demonstrate its capabilities in visualizing the Mn-labeled CST in addition to the 3D GE imaging.
The MRI scans were performed 24 hr after the Mn-delivery and electrical stimulation. The rat was anesthetized using spontaneous inhalation of 4% isoflurane for induction, followed by a mixture of 1.5% isoflurane, 30% oxygen, and air delivered through a nose mask. The head was stabilized on a Plexiglas holder and positioned in a 6 cm inner diameter volume coil for MRI scanning on a 9.4 T horizontal Varian scanner (Varian Inc., Palo Alto, CA). While in the scanner, the physiological condition of the rat was monitored using ECG, respiratory and temperature probes connected to a MR-compatible small animal monitoring and gating system (Model 1025, SA Instruments, Inc., Stony Brook, NY). The body temperature was maintained at 37°C by circulating warm air with 40 % humidity using a 5 cm diameter plastic tube fitted to the back door of the magnet bore.
After confirming the placement of the animal in the magnet's isocenter with scout images, T 1-weighted volumetric images covering the brain and SC at the cervical and thoracic levels were acquired using a 3D GE sequence (T R/T E = 45/4 ms and flip angle (FA) = 45°. The data were sampled on a matrix = 128 × 128 × 64 ranging over a volume of 70 × 32 × 32 mm3, and processed and interpolated to 256 × 256 × 128 pixels for the final display. Maximum intensity projections were generated to delineate the Mn enhancement in the CST over a desired thickness in the sagittal plane. Then, a sagittal IR-MEI was acquired (T R/T E/T I = 2000/12/550 ms, field-of-view (FOV) = 70 × 32 mm2, image matrix = 256 × 128, slice thickness = 2 mm and NEX = 4). Finally, axial IR-MEI was acquired (T R/T E/T I = 2000/17/550 ms, field-of-view (FOV) = 22 × 22 mm2, image matrix = 128 × 128, slice thickness = 2 mm and NEX = 4). The rat was then removed from the scanner, euthanized using cardiac puncture and the vertebral body was dissected from the animal. The excised sample with intact spine was scanned ex vivo at room temperature using an inductively coupled surface coil centered at the injury epicenter [10, 12]. High resolution multi-slice SE images were acquired in sagittal and axial planes (sagittal parameters: T R/T E = 2500/12 ms, field-of-view = 32 × 10 mm2, image matrix = 256 × 128, slice thickness = 0.5 mm and NEX = 2; axial parameters: T R/T E = 2500/12 ms, field-of-view = 10 × 10 mm2, image matrix = 128 × 128, slice thickness = 2 mm and NEX = 2). Finally, axial IR-MEI of the excised spine and SC were acquired (T R/T E/T I = 2000/15/550 ms, field-of-view (FOV) = 10 × 10 mm2, image matrix = 128 × 128, slice thickness = 2 mm and NEX = 4).
Results and discussion
Measurements from Mn-labeled regions, above and below the lesion, and at the epicenter of the injury.
Above the lesion
Below the lesion
Trace/3 (× 10-3 mm2/s)
0.69 ± 0.05
0.59 ± 0.07
0.64 ± 0.06
0.88 ± 0.08
0.79 ± 0.13
0.85 ± 0.10
The use of IR imaging produces tissue contrast variations dependent on the inversion time (TI). In IR acquisitions, the slight differences between the longitudinal relaxation times (T1) in the GM and the WM (including the CST) can be utilized to visualize the GM tissue as hypointense compared to the WM and CST, unlike the image contrast typically seen in the SE images of the SC in Fig. 3. The accumulation of Mn lowers the T1 in CST, introducing a new contrast behavior seen in the IR image of the SC. We determined that our acquisition parameters provided a good balance between image contrast (enhanced CST versus GM and WM signal suppression) and acquisition time at 9.4 T. Previous in vivo IR studies of the songbird brain at 7 T employed a somewhat different parameter set (T R/T E/T I = 4000/20/855 ms) . However, we note the variations in the species and organs studied, the experimental protocols followed and the magnetic field strength, each of which contributes to the differences in imaging parameters.
It is also important to note that we have obtained high resolution images using an inductively-coupled surface coil. This coil inherently produces an inhomogeneous rf field which ultimately yields spatially variant inversion. We notice that this may be an issue in the IR-MEI acquisition and lead to an incomplete suppression of the background tissue during the inversion recovery unless the power of the 90° rf pulse is adjusted carefully to focus on the SC.
This paper outlines the procedures required for successfully tracing the CST using MEI and demonstrates the potential of MEI in indirectly assessing the axonal fiber connectivity in injured rat SC. Further work is, however, warranted to confirm the reliability of tract tracing based on MEI using gold-standard histological analysis such as using transgenic  or anterograde tracer labeling approaches . Once the veracity of the methods is confirmed, future studies aiming to address different issues such as the ability to examine the CST connectivity in more severe injuries or to explore the role of dynamic imaging of intensity build-up in the CST below the injury with time will be possible. The data from such studies may be used to assess the density and distribution of the continuous fibers or to evaluate the efficacy of promoting the fiber connectivity in injured SC with endogeneous or exogeneous interventions.
This work was supported by NIH Grants NS052610 and NS054019. The author thanks Dr. Yong-Yue He for his help in animal preparation and surgeries, Dr. Baraa Al-Hafez for his help in MRI scans, Rebecca Chambers for her help in producing Fig. 1 and Josh Powell for his editorial help. The author also greatly appreciates Dr. William M. Brooks for critically reading the manuscript and providing his feedback.
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