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Use of a clinical MR scanner for imaging the rat brain
Brain Research Builrrin. Vol. 3 I. pp. 1 15-l LO, 1993 Printed in the USA. All
rights reserved.
Copyright
0363-9230/93 $6.00 + .oO r? 1993 Pergamon Press L Id.
Use of a Clinical MR Spanned for Imaging the Rat Brain DONALD
A. SMITH,* LAURENCE P. CLARKE,? JEFFREY A. FfEDLER,f- F. REED ~URTAG~.~ EUGENE A. BONAROTI,~ GREGORY J. SENGSTOCK~ AND GARY W. ARENDASH*~’
Received 12 May 1992; Accepted 4 August 1992 SMITH, D. A.. L. I? CLARKE, J. A. FIEDLER, F. R. MIJRTAGH, E. A. BONAROTI, G. J. SENGSTOCK AND G. W. ARENDASH. Use clf‘u clinical MR scunner,li,r imaging rhcl rat brain. BRAIN RES BULL 31(I/2)II5-120,1993.--Magnetic resonance imaging (MRI) and magnetic resonance spectroscopy are established techniques that enable noninvasive anatomic and functional tissue cham~e~~tion in vivo. These tools have been employed to pro~ex~~men~l models of neoplasia. cerebrovaxular disease, brain injury, and neurotransplantation in small animals. To date, these studies have been executed primarity on researchdedicated instruments of limited availability or resolution. Lking relatively stmi~tfo~ard software and hardware modi~cations ofa widely used clinical MRI unit. we were able to image numerous structures within the living rat brain incIuding the neost~atum, hippocampus. periaqueductal gray. and the ventricular system. Illustrative applications of this imaging technique in two intracerebral infusion models involving rats are presented. Such adaptation of clinical MRI scanners has the potential to signi~~antl~ expand the availability of high resolution in viva imaging of small animals for a variety of experimental protocols. Magnetic resonance imaging
Clinical scanner
Head coil
RODENT models are the current mainstays in neurobiologieal research. In vivo monito~ng of their neurochemicai~ metabolic, and electro~hysiolo~~c attributes within some experimental contexts is now possible. Until recently, however, anatomica analysis has relied on the examination of postmortem tissues. A method enabling ongoing anatomic surveillance of the nervous system has obvious applicability to numerous rodent models of neuro-clfogicdisease and injury. The living rat brain has been imaged using ~u~ose-dedi~ted/smal~ bore nuclear magnetic resonance systems (4.6,10,12-18,20). This equipment is very expensive, is not readiIy accessible to the research community, and has not always yielded qualitatively useful information; moreover, it may require very extended scanning times. Atthough clinical MRI units are widely available, in their standard configurations they do not provide sufficient spatial resolution to be useful in most research contexts due to signal-to-noise considerations. For example, the pixel size in routine human brain imaging is approximately 1.2 mm X 1.2 mm, assuming a 300 mm field of view (FOV) and a 2S6 X 256 acquisition matrix. This does not permit adequate resolution ofsubcortical structures within the rat brain, which itself has a mean diameter of approximately I2 mm.
Rat
The spatial resolution for a given instrument relates to the size of the sample voiume (voxel) from which the image is assembled. images assembled from voxels of smaller size will have greater spatial resolution than those assembled from larger voxeis. VOX& can be made smaller in the X-Y (axial) plane by shrinking the FOV, and can be made smaller in the Z dimension by reducing slice thickness” An initial strategy for enhancement of spatial resolution, therefore, includes console program selections for the smallest feasible FOV and stice thickness. As the voxel size is reduced, however, the magnitude of the radiofrequency (RF) signal emanating from the now smaller sampie volume at the resonant frequency is also diminished. This degrades the signal-to-noise ratio (SNR), which is the drawback of reduced voxel size. This problem can be overcome in part by applying signal-averaging techniques over multiple RF excitations. The resultant prolongation of scan time and the increased potential for movement artifacts place practical limits on the exercise of this strategy. Another method for improving SNR relies on a detector coil with a large fili factor. Because signal strength falls off inversely with the square of the distance from its source, SNR can be enhanced by reducing the distance to the receiving coil. This
’ To whom requests for reprints should be addressed.
115
distance would be minimized by using a detector coil whose bore is nearly filled by the study subject. Because even minute degrees of motion will seriously degrade image quality, some method to achieve rigid fixation of the rat’s head would also be highly desirable. Through the use of a specially fabricated 5 cm diameter RF antenna to enhance SNR and an integrated PlexigIas head holder to minimize movement artifacts, we have been able to modify a clinical MR unit (Siemens Magnetom, 1.0 Tesla) to enable excellent anatomic depiction of the living rodent brain. Utilizing various pulse sequences, representative axial plane images acquired in vivo from rats given intracerebral infusions of 6-hydroxydopamine or iron citrate are presented. The minute neuroanatomical structures demonstrated testify to the resolving capacity and usefulness of this system, which is made possible by relatively st~ightfo~ard software and hardware modifications. Unlike some published reports on rodent imaging (6). the scan protocols to be described can be executed in approximately 1 h. This time frame is relevant, both in terms of the expected duration of an anesthetic and because these machines will frequently share a clinical commitment. METHOD
All scanning was performed on a Siemens Magnetom 1.O T system that used linear polarized RF coils. .A double loop cylindrical detector coil was fabricated using 0.635 cm 3MTM copper seif-adhesing tape applied to a 5.0 cm (o.d.) by 6 cm acrylic tube (Fig. 1). The inner and outer loops of the coil were centered according to the design criteria of Joseph et al. (5,6). The coil was mounted on an acrylic cradle and contained a double row of holes to permit rigid fixation of the rat’s head by Plexiglas ear bars and wooden snout bars. The coil was tuned to a resonant frequency of 42 MHz through the addition of I85 pf capacitance to each half saddle. Axial images were acquired using a spin echo (SE) technique. The field gradient strength was set to 6.0 mT/m. enabling use of a slice thickness of 2.5 mm. The FOV was set to 50 mm. With a 256 X 256 image matrix, the computed image plane resolution was 0.1953 mm. T,-weighted images with TR = 0.7 sand TE = 23 ms at a slice gap of 2.5 mm were acquired. Eight RF excitations were employed and summed for signal averaging to reduce SNR. Total T, imaging time was approximately 24 min. T,-weighted images were acquired with TR = 2.3 s and TE = 80 ms, also with a slice gap of 2.5 mm and eight excitations. Imaging for Tz-weighted scans was approximately 48 min. 6-ti,vdro.qdopamine
infusions
Two female Sprague-Dawley rats received a stereotaxic infusion of 6-hydroxydopamine HCI (GOHDA; 8 rg/4 ~1 saline) into the right medial forebrain bundle 7 months prior to imaging. At infusion, animals weighed 258 and 262 g. With the incisor bar at 5” below horizontal, infusion coordinates were anterior (to the interaural line) 3.6 mm. lateral (from midline) 1.1 mm, and vertical (from the brain’s dorsal surface) 7.9 mm. Apomorphine-induced (25 mg/kg, SC) rotation behavior was tested over a 30-min period at monthly intervals until the time of scanning. At that time, animals were anesthetized with sodium pentobarbital (35 mg/kg, IP), affixed into the imaging coil, and Ti- and Tz-weighted axial images obtained as previously described. Following MRI scanning, the animals were sacriticed with an anesthetic overdose of sodium ~nto~rbi~i and perfused intracar&ally with 4% neutral buffered formalin. Brains were then removed and fixed for several days prior to taking 100 pm coronal sections on a freezing microtome. Whole brain sections
FIG. 1. (A) The double loop 5 cm diameter saddle coli used m the present study for MR signal detection. (B) An anesthetized rat secured by the Plexiglas head holder, which was itself integrated within the detector coil.
were then mounted and stained with thionin. Representative sections corresponding to the axial MRI scans were selected for reference and correlation. Iron Citrate Infusions Five male Sprague-Dawley rats (290-310 g) received two unilateral stereotaxic infusions of iron citrate solution (0.63 nmol iron chloride in 0.25 ~1of a ~t~te~i~r~nate vehicle) or vehicle solution into the substantia nigra beginning 1 month prior to imaging. Vehicle solution contained 46 mM sodium citrate dihydrate, 1,13 mM sodium bicarbonate, and 0.455 M Tris-HCI buffer in 0.07 1 M saline. The iron citrate infusate solution was adjusted to a pH of 7.4. Infusion coordinates were anterior 2.9 mm, lateral 2.3 mm, and vertical 7.5 mm. One week after initial nigral infusion, an identical infusion was made. On the day of imaging, anesthesia was induced with sodium pentobarbital(50 mg/kg, IP) and ?;-weighted axial images were obtained as previously described. Animals were sub~qnently sacrificed by anesthetic overdose; brains were removed, imme~on-fix~, histologically sectioned at 40 pm on a freezing mictrotome, and mounted on slides. Alternate brain sections were stained for thionin or for ferric iron (with DA3 intensification) according
MR IMAGING
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BRAIN
FIG. 2. Paired T,-weighted and T,-weighted MR scans at both thalamic (A.C) and midbrain (B,D) coronal levels from an animal that received an infusion of 6-OHDA into the right medial forebrain bundle 7 months prior to imaging. T,-weighted scans (A,B) and TLweighted scans (C.D) were obtained during the same session. Correlative histological sections from the same animal are presented at thalamic (E) and midbrain (F) levels. Abbreviations: CA, cerebral aqueduct: CC. corpus callosum; dorsal hippocampus: EC, entorhinal cortex: H. hy~th~amus; HC, hip~ampus; IP, inte~duncular nucleus; IPC inte~duncu~ar cistern: LV, lateral ventricle; NC. neocortex: OT. optic tract: P. pituitary gland; PAG, periaqueductai gray; POC, posterior neocortex: QC. quad~gem~nal cistern; SCO. superior colliculus: SN. substantia nigra: T. thalamus: WC, ventral hippocampus; IIIV. third ventricle.
to the methodology were counterstained
of Hill and Switzer (5); iron-stained with Mayer’s acid hemalum.
sections
RESULTS
Both 6-OHDA-infused animals had identical MR scans; paired axial T,- and T,-weighted images from one of these subjects are shown in Fig. 2. Images presented are those ob-
tained at the level of the thalamus and upper midbrain with correlative whole brain histologic sections. A variety of intraand extraaxial structures are identified and labeled in this &OHDA-infused rat. Neocortex and large subcortical structures such as the thalamus and hippocampus are readily recognized in topographic relationship to the corpus cailosum, the ventricular system. and the subarachnoid space. These relationships are made most obvious in the Tz-weighted imaging which
118
FIG. 3. (A,B) Tz-weighted MR scans at midbrain level from two rats at I month following either vehicle (A) or iron citrate (B) infusions into the substantia nigra. Note increased signal intensity (bright focal region) within the right substantia nigra of iron citrate-infused animal (B) and no change in nigral signal intensity as a result of vehicle infusion into the right substantia nigra (A). Shown in C is a correlative histological section through the rostra1 substantia nigra from the iron citrate-infused rat scanned above. Arrows indicate extent of nigral neuronal loss and gliosis (X20).
heightens internal tissue contrasts, rendering cerebrospinal tluid (CSF) bright white (long Tz relaxation time) and heavily myelinated white matter tracks black (short T> relaxation time). In addition, individual nuclei, such as the thalamus, may have sufficiently distinctive relaxation characteristics to enable their separate recognition in the absence of boundary structures. such as the corpus callosum (Fig. 2C). In T,-weighted imaging, CSF-containing spaces are visible as pure black (long T, relaxation time; Fig. 2A and B). As compared to Tz-weighted imaging, however, there is a lesser degree of internal tissue contrast to enable differentiation of separate nuclear groups or white matter tracts. For both 6-OHDA infused animals, neither T,- or Tz-weighted imaging revealed the site of neurotoxin infusion into the MFB. When comparing the substantia nigra and neostriatum ipsilateral vs. contralateral to neurotoxin infusion, no MRI signal differences were appreciated. Despite this, histological examination of thionin-stained brain sections rcvealed a near complete elimination of neurons in zona compacta of the substantia nigra ipsilateral to infusion (Fig. 2C). Over the entire 6-month period of testing for apomorphine-induced rotational behavior, the two 6-OHDA-infused subjects averaged 13.3 and 18.1 turns/min contralaterally, further substantiating the completeness of their neurotoxic lesions in destroying the vast majority of nigrostriatal dopaminergic neurons ipsilateral to infusion. Images of the cerebral aqueduct are of particular note. The diameter of the cerebral aqueduct in the fixed rat brain is approximately 0.3 mm (1 1). Its diameter in vivo is likely somewhat smaller. The demonstrated ability to visualize this structure correlates well with the calculated value of 0.195 mm for image plane resolution theoretically achieved by this system. It also underscores the important contributions of both spatial and contrast resolution to overall image quality. The cerebral aqueduct is clearly visible not only because the system can resolve a structure of this size, but because it contains CSF. which has a markedly different relaxation behavior compared to the surrounding brain parenchyma. This results in high image contrast at this interface and facilitates the detection of anatomic borders. All animals receiving intranigral iron citrate infusions exhibited similar scans at 1 month postinfusion. Figure 3B depict T?weighted axial scans from an iron citrate-infused subject through the level of the midbrain; this level corresponds closely to the scan level in Fig. 2B, D. and F. The site of iron citrate infusion is readily recognized as a focal bright region (long TZ relaxation time) within the substantia n&a. Because T,-weighted images from animals given vehicle infusions into the substantia nigra did not show the increase in nigral signal intensity characteristic of iron citrate-infused rats (Fig. 3A), the profound TZ relaxation time lengthening at the infusion site of iron citrate-infused animals may be attributable either to the paramagnetic effects of free or phagocytized iron and/or to highly localized injury and resultant edema. Histologic examination of the nigral infusion site from iron citrate-infused rats (Fig. 3C) could not differentiate between these possibilites because: a) extensive neuronal losses within and limited to the infused substantia nigra were observed. b) reactive glial cells in the substantia nigra were heavily embedded with iron, and c) a degree of edemic cavitation was usually seen. Consistent with a direct contribution from intranigrally infused iron in the increased nigral signal intensity observed are T2 scans of iron citrate and vehicle solutions alone. Compared to a relatively low signal intensity emanating from vehicle solution, iron citrate infusate exhibited a substantially increased signal intensity.
MR IMAGING
OF RAT
119
BRAIN
DISCUSSION
The present study indicates that relatively inexpensive adaptation of clinical MR scanners can achieve high resolution in vivo images of the rodent brain that are pertinent to a wide variety of experimental protocols. These adaptations include use of a small bore detector coil (to improve SNR) in conjunction with an integrated Plexiglas head holder (to eliminate animal movement artifacts). Numerous advantages make this adapted MR system attractive to basic researchers seeking to noninvasively characterize rodent brain tissues in vivo. Firstly, the wide availability of clinical MR units and the minimal adaptation costs involved make such MR imaging possible at a great many facilities. Secondly. in contrast to some research units requiring 3 or more h of scan time and animal anesthesia, the presently described unit can provide a contiguous series of high resolution T, images within I h. Thirdly, multiple scans of the same rat over time are facilitated by this adapted clinical unit. We have, in fact. scanned the same living animals on three separate occasions. Most studies involving MR imaging ofthe rodent brain have utilized research-based MR systems to provide images of normal brain (hf. to detect intracerebral infarctions/gliomas (3.4.10.11.14.16.18.20). or to investigate damage resulting from intracerebral neurotoxin infusions ( 15,17). Compared to the brain images obtained in these studies, the scans currently presented from a clinical MR scanner demonstrate a comparable or superior resolution of most neuroanatomical structures. The successful localization of a focal. small-volume iron citrate infusion within the substantia nigra of a living animal demonstrates the high resolution capacity of this adapted clinical unit. Such discrete detection of experimental brain manipulations clearly suggests use of this adapted clinical system in a wide variety of animal models related to degenerative brain diseases or brain injury. Although several previous studies have utilized clinical MR units to image the rat brain (1,2,19,21). the MR scans achieved in the present study appear superior in both contrast and resolution to scans presented in such studies: this, despite the fact that these clinical MR-based studies all involved pretreatment of animals with gadolinium to enhance image contrast. At 7 months following h-OHDA neurotoxic lesioning of the nigrostriatal dopaminergic pathway. no changes in Tz signal intensity were observed within the ipsilateral substantia nigra de-
spite massive histologically verified neuronal losses within zona compacta of substantia nigra. Moreover, MR scans at I. 2, and 4 months following identical 6-OHDA lesions in other animals also failed to show any changes in Tz signal intensity within the Iesioned substantia nigra (as observed by the investigators). These findings are consistent with our inability to visualize ibotenic acid-induced lesions of the basal forebrain’s nucleus basalis at various time points (22). Together. the above data strongly suggest that neurotoxic lesions within the rat brain do not produce tissue (i.e., intraparenchymal) changes that are readily imaged by current MRI technology. This conclusion is in contrast to a recent report that provided evidence that kainic acid-induced lesions ofthe rat neost~atuni are visualized as areas of increased signal intensity in TZ-weighted images ( I7). MR imaging is rapidly evolving. and several strategies exist for improving upon the results demonstrated here. Image quality and SNR could be further enhanced if the data were acquired on a higher field strength system with an appropriately tuned multielement quadrature coil; steeper field gradients would also enable reduced slice thickness and alternative imaging parameters should be further explored to determine optimal pulse sequences. ~ramagnetic contrast agents may also prove to be useful adjuncts ( I ,2. lh. I&19.2 I 1. The application of three-dimensional volume acquisition imaging methods would permit computation ofthinner slices without loss in SNR. Similarly, it should be possible to use surface-rendering techniques and image segmentation to displav these internal structures in three dimensions (9). Modifica;ions of this coil design are required for the more recent quadrature or circularly polarized RF coils that require active electronic decoupling methods. In summary. high resolution MR images of the living rat brain were obtained using a widely available clinical scanner and an easily fabricated small bore RF coil. This technique could be easily generalized to other animal models of neoplasia. cerehrovascular disease. brain injury. and neurotransplantation development.
ACKNOWLEDGEMENTS
This work was supported in part by a Grant from the United Parkinson’s Association (G.W.A.) and a lJSF Department of Surgery Research Grant (D.4.S.).
REFERENCES I
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5. 6. 7.
Baba. T.: Moriguchi. M.: Natori. Y.: Katsuki, C.; Inoue, T.: Fukui, M. Magnetic resonance imaging of experimental rat brain tumors: Histopathoiogical evaluation. Surg. Neural. 34:378-382: 1990. Bernstein. M.: Marotta. T.: Stewart, P.: Glen, J.: Resch. L.: Henkelman. M. Brain damage from “‘1 b~eh~ltherapy evaluated by MR imaging, a blood-brain harrier tracer. and light and electron microscopy in a rat model. J. Neurosurg. 73:585-593: 1990. Bradley, R.: Keng, ‘I’.:Eisenberg, H.: Quast, M.: Ward, G.: Campbell. G.: Hillman, G. Middle cerebral artery occlusion in rats studied by magnetic resonance imaging. Stroke 20: 1032-1036: 1989. Germane. 1.: Pitts. I-.; Berry, 1.: Moseley, M. Magnetic resonance imaging and “P magnetic resonance spectroscopy for evaluating focal cerebral ischemia. J. Neurosurg. 70:6 12-618: 1989. Hill, J.: SwitLer, R., Ill. The regional dist~bution and cellular localization of iron in the rat brain. Neuroscience 11:595-603: 1984. Johnson. G.: Thompson. M.; Drayer, B. Three-dimentional MRI microscopy of the normal rat brain. Magn. Reson. Med. 435 l-365: 1987. Joseph, P.; Fishman J. Design and evaluation of a radiofrequency coil for nuclear magnetic resonance imaging of fluorine and protons. Med. Phys. I2:679-683: 1985.
8. Joseph. P.: Summers. R. Coil design for short echo time sodium-23 nuclear magnetic resonance imaging. 1. Magn. Reson, 69:l98-205: 1986. 9. Jungke. M.; Van Se&n. W.: Bielke, G. In~orrna~iL~nprocessing in nuclear magnetic resonance imaging. Magn. Reson. Imaging h:6836Y3; 1988. IO. Knight, R.: Ordidge. R.: Helpern. J.: Chopp, M.; Rodolosi. L.: Peck, D. Temporal evolution of ischemic damage in rat brain measured by proton nuclear manetic resonance imaging. Stroke 22:802-808: 1991. II. Konig. J.; Klippel. A. The rat brain: A stereotaxic atlas ofthe forehrain and lower parts of the brain stem. Baftimore: William and Wilkins: 1963. 12. Lesiuk. H.: Sutherland. G.; Peeling J.: Butler. K.; Saunders. J. Effect of U74006F on Forebrain &hernia in rats. Stroke 22896-90 1: 1991. 13. London, R.; Toney, G.; Gahel. S.; Funk. A. Magnetic resonance imaging studies of the brains of anesthetized rats treated with manganese chloride. Brain Res. Bull. 23:229-235: 1989. 14. Mintorovitch, J.: Moseley, M.: Chileuitt. L.: Shimizu. H.; Cohen. Y.: Weinstein. P. Comparison of diffusion- and Z-weighted MRI
for the early detection ofcerebralischemia and reperfusion in rats. Magn. Res. Med. I8:39-50: 199 I. 15. Norman, A.; Thomas. S.; Pratt. R.; Samaratunga, R.: Sanberg, P. Magnetic resonance imaging of rat brain following kainic acid-induced lesions and fetal striatal tissue transplants. Brain Res. 483: 188-191; 1989. 16. Norman, A.: Thomas, S.; Pratt, R.; Samaratunga. R.; Sanberg, P. A manetic resonance imaging contrast agent differentiates between the vascular properties of fetal striatal tissue transplants and gliomas in rat brain in vivo. Brain Res. 503: 156-l 59: 1989. 17. Norman, A.; Thomas, S.; Pratt, R.: Samaratunga, R.: Sanberg, P. T, and Tz weighted magnetic resonance imaging ofexcitotoxin lesions and neural transplants in rat brain in vivo. Exp. Neural. 109: 164170: 1990.
18. Norman, A.; Bertram, K.: Thomas. S.; Pratt. K.; Samaratunga, K..
19.
20. 21.
22.
Sanberg. P. Magnetic resonance imaging of rat brain following m vivo disruption ofthe cerebral vasculature. Brain Res. Bull. 26:59i-. 597: 1991. Runge. V.; Jacobson, S.: Wood, M.; Kaufman. I).; Adelman. 1 MR imaging of rat brain glioma: Cd-DTPA versus Gd-DOTA. Radiology 166:835-838; 1988. Sauter, A.; Rudin, M.; Calcium antagonists for reduction ofbram damage in stroke. J. Cardiovasc. Pharmacol. I S(Supp. l):s43-S47:1990. Schmiedl, U.; Maravilla, K.: Nelson, J. Improved method for III v ivo magnetic resonance contrast media research. Invest. Radiol. 26~65-70; I99 I Smith. D.; Clarke, L.; Arendash, G. Magnetic resonance imaging ot ferrite-labeled neurotransplants. Sot. Neurosci. .Abstr. 17:I 142: 1988.
rights reserved.
Copyright
0363-9230/93 $6.00 + .oO r? 1993 Pergamon Press L Id.
Use of a Clinical MR Spanned for Imaging the Rat Brain DONALD
A. SMITH,* LAURENCE P. CLARKE,? JEFFREY A. FfEDLER,f- F. REED ~URTAG~.~ EUGENE A. BONAROTI,~ GREGORY J. SENGSTOCK~ AND GARY W. ARENDASH*~’
Received 12 May 1992; Accepted 4 August 1992 SMITH, D. A.. L. I? CLARKE, J. A. FIEDLER, F. R. MIJRTAGH, E. A. BONAROTI, G. J. SENGSTOCK AND G. W. ARENDASH. Use clf‘u clinical MR scunner,li,r imaging rhcl rat brain. BRAIN RES BULL 31(I/2)II5-120,1993.--Magnetic resonance imaging (MRI) and magnetic resonance spectroscopy are established techniques that enable noninvasive anatomic and functional tissue cham~e~~tion in vivo. These tools have been employed to pro~ex~~men~l models of neoplasia. cerebrovaxular disease, brain injury, and neurotransplantation in small animals. To date, these studies have been executed primarity on researchdedicated instruments of limited availability or resolution. Lking relatively stmi~tfo~ard software and hardware modi~cations ofa widely used clinical MRI unit. we were able to image numerous structures within the living rat brain incIuding the neost~atum, hippocampus. periaqueductal gray. and the ventricular system. Illustrative applications of this imaging technique in two intracerebral infusion models involving rats are presented. Such adaptation of clinical MRI scanners has the potential to signi~~antl~ expand the availability of high resolution in viva imaging of small animals for a variety of experimental protocols. Magnetic resonance imaging
Clinical scanner
Head coil
RODENT models are the current mainstays in neurobiologieal research. In vivo monito~ng of their neurochemicai~ metabolic, and electro~hysiolo~~c attributes within some experimental contexts is now possible. Until recently, however, anatomica analysis has relied on the examination of postmortem tissues. A method enabling ongoing anatomic surveillance of the nervous system has obvious applicability to numerous rodent models of neuro-clfogicdisease and injury. The living rat brain has been imaged using ~u~ose-dedi~ted/smal~ bore nuclear magnetic resonance systems (4.6,10,12-18,20). This equipment is very expensive, is not readiIy accessible to the research community, and has not always yielded qualitatively useful information; moreover, it may require very extended scanning times. Atthough clinical MRI units are widely available, in their standard configurations they do not provide sufficient spatial resolution to be useful in most research contexts due to signal-to-noise considerations. For example, the pixel size in routine human brain imaging is approximately 1.2 mm X 1.2 mm, assuming a 300 mm field of view (FOV) and a 2S6 X 256 acquisition matrix. This does not permit adequate resolution ofsubcortical structures within the rat brain, which itself has a mean diameter of approximately I2 mm.
Rat
The spatial resolution for a given instrument relates to the size of the sample voiume (voxel) from which the image is assembled. images assembled from voxels of smaller size will have greater spatial resolution than those assembled from larger voxeis. VOX& can be made smaller in the X-Y (axial) plane by shrinking the FOV, and can be made smaller in the Z dimension by reducing slice thickness” An initial strategy for enhancement of spatial resolution, therefore, includes console program selections for the smallest feasible FOV and stice thickness. As the voxel size is reduced, however, the magnitude of the radiofrequency (RF) signal emanating from the now smaller sampie volume at the resonant frequency is also diminished. This degrades the signal-to-noise ratio (SNR), which is the drawback of reduced voxel size. This problem can be overcome in part by applying signal-averaging techniques over multiple RF excitations. The resultant prolongation of scan time and the increased potential for movement artifacts place practical limits on the exercise of this strategy. Another method for improving SNR relies on a detector coil with a large fili factor. Because signal strength falls off inversely with the square of the distance from its source, SNR can be enhanced by reducing the distance to the receiving coil. This
’ To whom requests for reprints should be addressed.
115
distance would be minimized by using a detector coil whose bore is nearly filled by the study subject. Because even minute degrees of motion will seriously degrade image quality, some method to achieve rigid fixation of the rat’s head would also be highly desirable. Through the use of a specially fabricated 5 cm diameter RF antenna to enhance SNR and an integrated PlexigIas head holder to minimize movement artifacts, we have been able to modify a clinical MR unit (Siemens Magnetom, 1.0 Tesla) to enable excellent anatomic depiction of the living rodent brain. Utilizing various pulse sequences, representative axial plane images acquired in vivo from rats given intracerebral infusions of 6-hydroxydopamine or iron citrate are presented. The minute neuroanatomical structures demonstrated testify to the resolving capacity and usefulness of this system, which is made possible by relatively st~ightfo~ard software and hardware modifications. Unlike some published reports on rodent imaging (6). the scan protocols to be described can be executed in approximately 1 h. This time frame is relevant, both in terms of the expected duration of an anesthetic and because these machines will frequently share a clinical commitment. METHOD
All scanning was performed on a Siemens Magnetom 1.O T system that used linear polarized RF coils. .A double loop cylindrical detector coil was fabricated using 0.635 cm 3MTM copper seif-adhesing tape applied to a 5.0 cm (o.d.) by 6 cm acrylic tube (Fig. 1). The inner and outer loops of the coil were centered according to the design criteria of Joseph et al. (5,6). The coil was mounted on an acrylic cradle and contained a double row of holes to permit rigid fixation of the rat’s head by Plexiglas ear bars and wooden snout bars. The coil was tuned to a resonant frequency of 42 MHz through the addition of I85 pf capacitance to each half saddle. Axial images were acquired using a spin echo (SE) technique. The field gradient strength was set to 6.0 mT/m. enabling use of a slice thickness of 2.5 mm. The FOV was set to 50 mm. With a 256 X 256 image matrix, the computed image plane resolution was 0.1953 mm. T,-weighted images with TR = 0.7 sand TE = 23 ms at a slice gap of 2.5 mm were acquired. Eight RF excitations were employed and summed for signal averaging to reduce SNR. Total T, imaging time was approximately 24 min. T,-weighted images were acquired with TR = 2.3 s and TE = 80 ms, also with a slice gap of 2.5 mm and eight excitations. Imaging for Tz-weighted scans was approximately 48 min. 6-ti,vdro.qdopamine
infusions
Two female Sprague-Dawley rats received a stereotaxic infusion of 6-hydroxydopamine HCI (GOHDA; 8 rg/4 ~1 saline) into the right medial forebrain bundle 7 months prior to imaging. At infusion, animals weighed 258 and 262 g. With the incisor bar at 5” below horizontal, infusion coordinates were anterior (to the interaural line) 3.6 mm. lateral (from midline) 1.1 mm, and vertical (from the brain’s dorsal surface) 7.9 mm. Apomorphine-induced (25 mg/kg, SC) rotation behavior was tested over a 30-min period at monthly intervals until the time of scanning. At that time, animals were anesthetized with sodium pentobarbital (35 mg/kg, IP), affixed into the imaging coil, and Ti- and Tz-weighted axial images obtained as previously described. Following MRI scanning, the animals were sacriticed with an anesthetic overdose of sodium ~nto~rbi~i and perfused intracar&ally with 4% neutral buffered formalin. Brains were then removed and fixed for several days prior to taking 100 pm coronal sections on a freezing microtome. Whole brain sections
FIG. 1. (A) The double loop 5 cm diameter saddle coli used m the present study for MR signal detection. (B) An anesthetized rat secured by the Plexiglas head holder, which was itself integrated within the detector coil.
were then mounted and stained with thionin. Representative sections corresponding to the axial MRI scans were selected for reference and correlation. Iron Citrate Infusions Five male Sprague-Dawley rats (290-310 g) received two unilateral stereotaxic infusions of iron citrate solution (0.63 nmol iron chloride in 0.25 ~1of a ~t~te~i~r~nate vehicle) or vehicle solution into the substantia nigra beginning 1 month prior to imaging. Vehicle solution contained 46 mM sodium citrate dihydrate, 1,13 mM sodium bicarbonate, and 0.455 M Tris-HCI buffer in 0.07 1 M saline. The iron citrate infusate solution was adjusted to a pH of 7.4. Infusion coordinates were anterior 2.9 mm, lateral 2.3 mm, and vertical 7.5 mm. One week after initial nigral infusion, an identical infusion was made. On the day of imaging, anesthesia was induced with sodium pentobarbital(50 mg/kg, IP) and ?;-weighted axial images were obtained as previously described. Animals were sub~qnently sacrificed by anesthetic overdose; brains were removed, imme~on-fix~, histologically sectioned at 40 pm on a freezing mictrotome, and mounted on slides. Alternate brain sections were stained for thionin or for ferric iron (with DA3 intensification) according
MR IMAGING
OF RAT
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BRAIN
FIG. 2. Paired T,-weighted and T,-weighted MR scans at both thalamic (A.C) and midbrain (B,D) coronal levels from an animal that received an infusion of 6-OHDA into the right medial forebrain bundle 7 months prior to imaging. T,-weighted scans (A,B) and TLweighted scans (C.D) were obtained during the same session. Correlative histological sections from the same animal are presented at thalamic (E) and midbrain (F) levels. Abbreviations: CA, cerebral aqueduct: CC. corpus callosum; dorsal hippocampus: EC, entorhinal cortex: H. hy~th~amus; HC, hip~ampus; IP, inte~duncular nucleus; IPC inte~duncu~ar cistern: LV, lateral ventricle; NC. neocortex: OT. optic tract: P. pituitary gland; PAG, periaqueductai gray; POC, posterior neocortex: QC. quad~gem~nal cistern; SCO. superior colliculus: SN. substantia nigra: T. thalamus: WC, ventral hippocampus; IIIV. third ventricle.
to the methodology were counterstained
of Hill and Switzer (5); iron-stained with Mayer’s acid hemalum.
sections
RESULTS
Both 6-OHDA-infused animals had identical MR scans; paired axial T,- and T,-weighted images from one of these subjects are shown in Fig. 2. Images presented are those ob-
tained at the level of the thalamus and upper midbrain with correlative whole brain histologic sections. A variety of intraand extraaxial structures are identified and labeled in this &OHDA-infused rat. Neocortex and large subcortical structures such as the thalamus and hippocampus are readily recognized in topographic relationship to the corpus cailosum, the ventricular system. and the subarachnoid space. These relationships are made most obvious in the Tz-weighted imaging which
118
FIG. 3. (A,B) Tz-weighted MR scans at midbrain level from two rats at I month following either vehicle (A) or iron citrate (B) infusions into the substantia nigra. Note increased signal intensity (bright focal region) within the right substantia nigra of iron citrate-infused animal (B) and no change in nigral signal intensity as a result of vehicle infusion into the right substantia nigra (A). Shown in C is a correlative histological section through the rostra1 substantia nigra from the iron citrate-infused rat scanned above. Arrows indicate extent of nigral neuronal loss and gliosis (X20).
heightens internal tissue contrasts, rendering cerebrospinal tluid (CSF) bright white (long Tz relaxation time) and heavily myelinated white matter tracks black (short T> relaxation time). In addition, individual nuclei, such as the thalamus, may have sufficiently distinctive relaxation characteristics to enable their separate recognition in the absence of boundary structures. such as the corpus callosum (Fig. 2C). In T,-weighted imaging, CSF-containing spaces are visible as pure black (long T, relaxation time; Fig. 2A and B). As compared to Tz-weighted imaging, however, there is a lesser degree of internal tissue contrast to enable differentiation of separate nuclear groups or white matter tracts. For both 6-OHDA infused animals, neither T,- or Tz-weighted imaging revealed the site of neurotoxin infusion into the MFB. When comparing the substantia nigra and neostriatum ipsilateral vs. contralateral to neurotoxin infusion, no MRI signal differences were appreciated. Despite this, histological examination of thionin-stained brain sections rcvealed a near complete elimination of neurons in zona compacta of the substantia nigra ipsilateral to infusion (Fig. 2C). Over the entire 6-month period of testing for apomorphine-induced rotational behavior, the two 6-OHDA-infused subjects averaged 13.3 and 18.1 turns/min contralaterally, further substantiating the completeness of their neurotoxic lesions in destroying the vast majority of nigrostriatal dopaminergic neurons ipsilateral to infusion. Images of the cerebral aqueduct are of particular note. The diameter of the cerebral aqueduct in the fixed rat brain is approximately 0.3 mm (1 1). Its diameter in vivo is likely somewhat smaller. The demonstrated ability to visualize this structure correlates well with the calculated value of 0.195 mm for image plane resolution theoretically achieved by this system. It also underscores the important contributions of both spatial and contrast resolution to overall image quality. The cerebral aqueduct is clearly visible not only because the system can resolve a structure of this size, but because it contains CSF. which has a markedly different relaxation behavior compared to the surrounding brain parenchyma. This results in high image contrast at this interface and facilitates the detection of anatomic borders. All animals receiving intranigral iron citrate infusions exhibited similar scans at 1 month postinfusion. Figure 3B depict T?weighted axial scans from an iron citrate-infused subject through the level of the midbrain; this level corresponds closely to the scan level in Fig. 2B, D. and F. The site of iron citrate infusion is readily recognized as a focal bright region (long TZ relaxation time) within the substantia n&a. Because T,-weighted images from animals given vehicle infusions into the substantia nigra did not show the increase in nigral signal intensity characteristic of iron citrate-infused rats (Fig. 3A), the profound TZ relaxation time lengthening at the infusion site of iron citrate-infused animals may be attributable either to the paramagnetic effects of free or phagocytized iron and/or to highly localized injury and resultant edema. Histologic examination of the nigral infusion site from iron citrate-infused rats (Fig. 3C) could not differentiate between these possibilites because: a) extensive neuronal losses within and limited to the infused substantia nigra were observed. b) reactive glial cells in the substantia nigra were heavily embedded with iron, and c) a degree of edemic cavitation was usually seen. Consistent with a direct contribution from intranigrally infused iron in the increased nigral signal intensity observed are T2 scans of iron citrate and vehicle solutions alone. Compared to a relatively low signal intensity emanating from vehicle solution, iron citrate infusate exhibited a substantially increased signal intensity.
MR IMAGING
OF RAT
119
BRAIN
DISCUSSION
The present study indicates that relatively inexpensive adaptation of clinical MR scanners can achieve high resolution in vivo images of the rodent brain that are pertinent to a wide variety of experimental protocols. These adaptations include use of a small bore detector coil (to improve SNR) in conjunction with an integrated Plexiglas head holder (to eliminate animal movement artifacts). Numerous advantages make this adapted MR system attractive to basic researchers seeking to noninvasively characterize rodent brain tissues in vivo. Firstly, the wide availability of clinical MR units and the minimal adaptation costs involved make such MR imaging possible at a great many facilities. Secondly. in contrast to some research units requiring 3 or more h of scan time and animal anesthesia, the presently described unit can provide a contiguous series of high resolution T, images within I h. Thirdly, multiple scans of the same rat over time are facilitated by this adapted clinical unit. We have, in fact. scanned the same living animals on three separate occasions. Most studies involving MR imaging ofthe rodent brain have utilized research-based MR systems to provide images of normal brain (hf. to detect intracerebral infarctions/gliomas (3.4.10.11.14.16.18.20). or to investigate damage resulting from intracerebral neurotoxin infusions ( 15,17). Compared to the brain images obtained in these studies, the scans currently presented from a clinical MR scanner demonstrate a comparable or superior resolution of most neuroanatomical structures. The successful localization of a focal. small-volume iron citrate infusion within the substantia nigra of a living animal demonstrates the high resolution capacity of this adapted clinical unit. Such discrete detection of experimental brain manipulations clearly suggests use of this adapted clinical system in a wide variety of animal models related to degenerative brain diseases or brain injury. Although several previous studies have utilized clinical MR units to image the rat brain (1,2,19,21). the MR scans achieved in the present study appear superior in both contrast and resolution to scans presented in such studies: this, despite the fact that these clinical MR-based studies all involved pretreatment of animals with gadolinium to enhance image contrast. At 7 months following h-OHDA neurotoxic lesioning of the nigrostriatal dopaminergic pathway. no changes in Tz signal intensity were observed within the ipsilateral substantia nigra de-
spite massive histologically verified neuronal losses within zona compacta of substantia nigra. Moreover, MR scans at I. 2, and 4 months following identical 6-OHDA lesions in other animals also failed to show any changes in Tz signal intensity within the Iesioned substantia nigra (as observed by the investigators). These findings are consistent with our inability to visualize ibotenic acid-induced lesions of the basal forebrain’s nucleus basalis at various time points (22). Together. the above data strongly suggest that neurotoxic lesions within the rat brain do not produce tissue (i.e., intraparenchymal) changes that are readily imaged by current MRI technology. This conclusion is in contrast to a recent report that provided evidence that kainic acid-induced lesions ofthe rat neost~atuni are visualized as areas of increased signal intensity in TZ-weighted images ( I7). MR imaging is rapidly evolving. and several strategies exist for improving upon the results demonstrated here. Image quality and SNR could be further enhanced if the data were acquired on a higher field strength system with an appropriately tuned multielement quadrature coil; steeper field gradients would also enable reduced slice thickness and alternative imaging parameters should be further explored to determine optimal pulse sequences. ~ramagnetic contrast agents may also prove to be useful adjuncts ( I ,2. lh. I&19.2 I 1. The application of three-dimensional volume acquisition imaging methods would permit computation ofthinner slices without loss in SNR. Similarly, it should be possible to use surface-rendering techniques and image segmentation to displav these internal structures in three dimensions (9). Modifica;ions of this coil design are required for the more recent quadrature or circularly polarized RF coils that require active electronic decoupling methods. In summary. high resolution MR images of the living rat brain were obtained using a widely available clinical scanner and an easily fabricated small bore RF coil. This technique could be easily generalized to other animal models of neoplasia. cerehrovascular disease. brain injury. and neurotransplantation development.
ACKNOWLEDGEMENTS
This work was supported in part by a Grant from the United Parkinson’s Association (G.W.A.) and a lJSF Department of Surgery Research Grant (D.4.S.).
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