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,
,,1
* Athinoula A. Martinos Center for Biomedical Imaging;
Gene Transcript Targeting and Repair Laboratory, Division of Neuroradiology;
Department of Radiology; and
Department of Pediatrics, Massachusetts General Hospital and Harvard Medical School, Charlestown, Massachusetts, USA
1Correspondence: 149 Thirteenth St., Rm. 2410, Charlestown, MA 02129, USA. E-mail: philipl{at}nmr.mgh.harvard.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: angiogenesis • antisense technology • blood-brain barrier • bulbar conjunctiva • gene expression • molecular imaging
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3
4
5)
. Detection of gliosis or cells of abnormal growth has traditionally involved immunohistochemistry methods to detect elevated antigen of glial fibrillary acidic protein (GFAP) levels in postmortem tissue samples (6
7
8)
. However, brain biopsy to acquire tissue for these conventional assays is by itself invasive, and the procedure often results in the removal of important tissue.
MRI is a powerful and noninvasive tool for in vivo imaging of soft tissue (9
10
11
12)
but is somewhat limited by the unavailability of suitable probes for the CNS. We have designed modular MR probes capable of reporting specific cells based on gene transcription in living brains of ordinary subjects (8
, 13
, 14)
. In several proof-of-concept studies, we have developed and validated the delivery and neuronal uptake of modular MR probes in the cerebrospinal fluid after intracerebroventricular (i.c.v.) infusion. Although this method of delivery is invasive, we have demonstrated its effectiveness for mRNA reporting and probe distribution through the Virchow-Robins space. Others have reported that the lymphatic system assists the circulation of peritoneal fluid (15)
and preferentially retains superparamagnetic iron oxide nanoparticles (SPION) (16)
. Lymphatic vessels are present in the eyelid and bulbar conjunctiva (17
18
19
20)
, and the lymphatic fluids eventually merge with circulating blood in the vena cava for redistribution in the arterial vessels. Given that MR contrast agent can be delivered to the brain cells via arterial injection when the BBB is disrupted (21)
, we hypothesize that an MR probe can be distributed via the lymphatic system, either by intraperitoneal (i.p.) injection or by eyedrop solution to the conjunctival sac, in C57BL6 mice that experience BBB leakage. Here, we demonstrate noninvasive delivery of MR contrast agent to the brains of live mice, using a modular MR probe that targets the gene transcript of GFAP in glia and astrocytes and a control complementary to the mRNA of β-actin. The ability to deliver an MR contrast probe noninvasively and to label cells based on their intracellular gene transcripts will open the door to myriad applications in the brain and throughout the body.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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General surgical preparation
Male C57BL6 mice (24±3 g; Taconic Farms, Germantown, NY, USA) were housed under diurnal lighting conditions and allowed food and tap water ad libitum. The mice were anesthetized with ketamine (80 mg/kg, i.p.) and xylazine (12 mg/kg, i.p.) prior to surgical procedures. We induced BBB leakage by one of three methods: by cortical spreading depression, by inflicting a puncture wound and administering an i.c.v. injection of 2 µl saline in a Hamilton syringe with a 26S gauge needle to the left cerebral ventricle (14)
, or by performing bilateral carotid artery occlusion (BCAO) for 60 min (8
, 13)
. Another group of animals underwent a sham operation (the same surgical procedure without the puncture wound or vessel occlusion for cerebral ischemia) at the same time. Animals that underwent the BCAO procedure typically experienced a 25% drop in body weight within the first 3 days and gradually recovered to pre-BCAO weight in 5 days (unpublished observations); we therefore delayed the probe delivery and procedures for gene activity assays using transcription MRI (tMRI) until 5 days after surgery.
Cortical spreading depression
Under isoflurane anesthesia (2.5% induction, 1% maintenance, in 70% N2O/30% O2), we used a slightly modified topical KCl application method (22
, 23)
to assess cortical spreading depression (CSD) susceptibility in spontaneously breathing mice. With the anesthetized mouse positioned in a stereotactic frame (David Kopf Instruments, Tujunga, CA, USA), we drilled 2 burr holes under saline cooling at the noted coordinates to apply KCl to the occipital cortex (anterior-posterior=–1.5 mm; left-right=2 mm from bregma; diameter=2 mm) and at recording site 1 (anterior-posterior=1 mm; left-right=–2 mm from bregma; diameter=1 mm). The dura was kept intact to minimize trauma. We placed a glass microelectrode at a depth of 300 µm to record extracellular steady potential (DC) and electrocorticogram (ECoG). The reference (Ag/AgCl) electrode was placed subcutaneously in the neck. After surgical preparation, we irrigated the cortex with saline for 20 min, allowing it to recover. A cotton ball (2-mm diameter) soaked with 300 mM KCl was placed on the dura to induce CSD, after which we gently washed the cortex with saline. After 8 min, we induced another CSD using the same method. A total of 3 CSDs were elicited in each mouse. Mice in the sham group underwent the same surgical procedure, using the same anesthesia protocol; however, the cortex was exposed to saline only, so as not to elicit CSD. Data were continuously recorded using a data acquisition system for off-line analysis (Power Lab Instruments, Colorado Springs, CO, USA). We measured the DC shift amplitude, duration at half-maximal amplitude, and maximum onset slope. Potassium chloride-evoked CSDs were detected by a characteristic slow DC potential shift and ECoG suppression.
Conjugation of biotinylated sODN to SPION-NA and immunohistochemistry
We synthesized 5'-biotin-labeled antisense phosphorothioate-modified oligodeoxynucleotides (sODNs) for glia and astrocytes (sODN-gfap; 5'-gtctccgctccatcctgccc-3') to GFAP mRNA of the mouse (24)
, using methods previously reported for sODN-cfos, sODN-β-actin, and sODN-Ran; the sequences of these probes have also been reported previously (8
, 13)
. We used phosphorothioate modification of all nucleotide bridges to protect the single-stranded ODNs from nonspecific nucleases, and the resulting sODNs were purified by polyacrylamide gel electrophoresis. For histological evidence of uptake, we labeled the sODNs with fluorescein isothiocyanate (FITC). We counterstained postmortem brain tissue with murine monoclonal antibodies against neurin- and Cy-3-labeled rabbit anti-mouse IgG to reduce green autofluorescence. We detected cells expressing GFAP or β-actin using rabbit IgG against GFAP or β-actin and Cy-3-labeled polyclonal anti-rabbit IgG (8
, 13)
, and cerebral vascular endothelia using Griffonia simplicifolia lectin 1-conjugated FITC (Vector Laboratories, Burlingame, CA, USA) (25)
.
Preparation of the SPION-sODN
Preparation of the probe complex and MRI acquisition were performed using methods previously described (8
, 13)
, except freshly synthesized SPION was functionalized with chloroethylamine (2 M) in 1.5N NaOH solution, and linked to NeutrAvidin (NA) in the presence of 1 M sodium cyanoborohydride (both from Pierce Biotechnology, Rockford, IL, USA). The resulting covalently linked product, SPION-NA, was filtered and dialyzed against a 20x volume of sodium citrate buffer solution (25 mM, pH 8.0), using a Centricon Plus-100 filter (100 kD cutoff, Millipore Corp., Bedford, MA, USA). The activated SPION (SPION-NA) was stored in an amber-colored bottle at 4°C, at a concentration of 3–4 mg Fe/ml sodium citrate buffer. We mixed 100 µl SPION-NA (3 mg Fe/ml or 9 nmol SPION/ml) with sODN or sODN-FITC (1.8 nmol) on ice for 1 h. Immediately before application, we added 4 µl lipofectamine 2000 (1 mg/ml; Invitrogen Life Sciences, Carlsbad, CA, USA). A total of 0.1 ml of the MR probe solution was i.p. injected into each mouse.
Ophthalmic route of delivery
We delivered SPION-sODN-FITC (4 mg Fe/kg in 0.1 ml and lipofectamine, 0.05 mg/ml) by eyedropper (10 µl every 10 min under ketamine (100 mg/kg) and xylazine (10 mg/kg) anesthesia i.p.).
MRI protocol
MRI assessment of SPION retention in the regions of interest (ROIs) had been reported (8
, 13)
. To evaluate the level of probe retention, we calculated the mean R2* values in the cortex, hippocampus, and striatum. The average R2* values and SEM from each of these groups were obtained and analyzed statistically (14)
. To identify ROI with cell expressing elevated GFAP or actin antigen (MR cell typing), we identified regions where SPION retention was significantly above the preinfusion baseline of the same mouse using R2* maps. The hotspots were identified by referencing the stereotactic coordinates of hippocampus in C57BL6 mouse brains (26)
and confirmed by immunohistochemistry examinations. We acquired diffusion-weighted MRI (DWI) with parameters as follows: TR/TE = 3000/25 ms, two b values of 154 and 1294 s/mm2 along the z direction, 8 repetitions, 
= 12 ms, 
= 8 ms, 180 x 180 µm2 in-plane resolution, and 1 mm slice thickness for assessment of tissue injury. Maps of the apparent diffusion coefficient (ADC) were calculated by fitting the DWI images to the equation M = Mo x exp (–b ADC) using MRVision (Winchester, MA, USA). For the detection of BBB leakage, animals were scanned before the injection of Gd-DTPA (Magnevist; Shering, Berlin, Germany) using T1-weighted three-dimensional spin echo images (TR/TE=400/11 ms, 120x120 µm2 in-plane resolution and 0.5 mm thickness, NA=2). Gd-DTPA was administered to the jugular vein (0.1 mM/kg) and mice were imaged within 10 min. Extensive areas of enhanced T1 signals compared to the pre-Gd scans were considered to be areas of the brain which were leaky.
Statistical analysis
We performed a power calculation on the data gathered from the first set of animals, using in-house software to calculate the number of animals required in each group to achieve 90% power for a P value of 0.01 (8
, 13)
. If the minimum number of animals calculated was greater than 15, we revised the hypothesis and the protocol. We repeated each study using at least one mouse more in each treatment group than indicated by the power calculation. After we had acquired data from the minimum number of animals, we computed the mean and SEM from the averaged values in each group and compared the statistical significance of these values using a t test (one-tail, type II or equal variant, GraphPad Prism IV, GraphPad Software, San Diego, CA, USA). A value of P < 0.05 was considered statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 13)
, suggesting the lymphatic system may participate in the distribution of MR probe in the brain. MR contrast agent can be delivered to the brain by intravascular injection when the BBB is disrupted (21)
. Knowing that neurological disorders induce BBB leakage and that the intraperitoneal fluid in the lymphatic vessels merges with blood in the vascular system, we hypothesized that our MR contrast probe could be effectively and noninvasively distributed in the living brains of C57BL6 mice if BBB leakage occurred after cerebral ischemia. Figure 1
A shows the protocol for inducing BBB leakage that mimics conditions of acute neurological disorders. Figure 1B
demonstrates that 60-min BCAO in one of three C57BL6 mice induces BBB leakage within 3 h of reperfusion in the cortex and striatum in both hemispheres. In a separate group of animals, we detected hyperintense DWI at 1 or 2 days after BCAO (Fig. 2
A); mice that underwent sham operation did not exhibit such hyperintense DWI (Fig. 2B
). Although hyperintense DWI is reversible, BBB leakage developed in the regions showing hyperintense DWI after BCAO in C57BL6 mice (Savitz et al., unpublished data).
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To test SPION-sODN delivery to the brain in subjects that experienced BBB leakage, we conjugated NA-labeled SPION to biotinylated and phosphorothioate-modified oligodeoxynucleotides (biosODN) complementary to c-fos mRNA (sODN-cfos) or with a random sequence (sODN-Ran, no cellular target). We delivered SPION-cfos (
10 mg Fe/kg) by i.p. injection 5 days later. We acquired T2* MRI the day after injection and, because R2* is positively correlated with intracellular iron oxide in the mouse brain, constructed R2* maps of SPION-cfos retention (14)
. As shown in Fig. 2C
, R2* levels in the striatum increased 1 day after delivery and then diminished 4 days later to a level not different from that measured prior to i.p. injection. We found significant retention of SPION-cfos in the striatum and cortex, but retention of SPION-Ran was not significant when compared to the baseline R2* maps (Fig. 2D
). We also tested the retention of an MR probe without linkage between sODN-cfos and SPION (SPION and sODN-cfos were mixed, but not linked); we observed no uptake.
These data suggest that the fluid in the peritoneal cavity most likely distributes SPION-sODN by virtue of the eventual merging of the lymphatic and vascular circulations, aiding the MR probe in reaching the brain cells through BBB leakage. Given that lymphatic vessels exist in the stroma under the bulbar conjunctiva (17
18
19
20)
, we chose to examine the feasibility of imaging and targeting intracellular mRNA using our novel contrast agent and MRI after administering the probe via an ophthalmic route of delivery (OTRD). We selected an MR probe targeting the mRNA of β-actin for the distribution assay because its transcripts are not drastically altered immediately after brain injury. We aimed to apply MRI with OTRD to evaluate probe delivery after cortical spreading depression (n=4 pairs), which has been shown to induce microleakage in the BBB (22)
. Figure 3
A shows SPION-β-actin retention in the brains of live mice 5 days after CSD episode. Retention of SPION–sODN was observed only for mice with CSD; no retention was observed in sham-operated mice. Figure 3C
shows subtraction R2* maps of one representative animal from 3A, revealing elevated R2* using a computer-generated pixel scale (0–150% with an increment of 25%). The ROI in the cortex, hippocampus, and striatum show a significant increase in R2* (t test, Fig. 3B
). Validating the retention of SPION–sODN, Fig. 3D
shows histological evidence of sODN–β-actin-FITC uptake in the neuron where β-actin is expressed. Probe uptake occurred in mice that underwent CSD induction, but not in those that had the sham operation. We have demonstrated a novel noninvasive technique for MRI and gene transcription in living animals.
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Having demonstrated the uptake of the SPION-β-actin probe in neurons after CSD, we chose to examine the feasibility of using OTRD and transcription MRI (tMRI) to target and image intracellular mRNA a few weeks after acute neurological disorder. We induced BBB leakage in the left (ipsilateral) hemisphere using i.c.v. puncture in six mice; 3 days later, we sacrificed three of the animals for histological examination of gliosis around the injured site. We found that all had gliosis around the injured site (arrow, Fig. 4
A). This model allows comparisons of injured ROI and noninjured hemispheres in the same brain. In the remaining three mice, we acquired T2-weighted MRI 8 wk after i.c.v. puncture and observed no remarkable lesion in the left cortex compared to the right, except possible water retention at the injured site (arrow, Fig. 4B
). We waited for 8 weeks after injury so as to avoid detecting reactive gliosis or enhanced macrophages at the injured site, which may elevate R2*. We made a short DNA sequence complementary to GFAP mRNA of the mouse (17
18
19
20)
and delivered the MR probe (SPION-gfap) in an eyedrop solution via the eyelid and bulbar conjunctiva. R2* maps 1 day after OTRD of SPION-gfap revealed regions with hyperintense R2* at the injury site in all three animals (arrows, Fig. 4C
). This hyperintense focal R2* region after SPION-gfap was consistent with immunohistochemistry results showing genuine gliosis (arrow, Fig. 4A
) and tangles of GFAP-positive cells in the tissue surrounding the wound in the ipsilateral cortex (asterisk, Fig. 4D
), but normal patterns of GFAP-positive cells in the contralateral hemisphere (Fig. 4E
). Hyperintense R2* maps were no longer detectable in MRI repeated 1 wk later. Our data presented here support our assertion that OTRD can effectively deliver SPION-sODN to the brain that experiences BBB disruption.
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To apply OTRD for detecting gliosis after neurological disorders, we investigated gliosis reporting using SPION-gfap in two groups of animals that had experienced either cerebral ischemia (n=7) or sham operation (n=4). We observed hyperintense DWI/ADC drop 1 day after cerebral ischemia in all seven of the ischemic mice; hyperintense DWI/ADC drop was bilateral in five of the mice, unilateral in two. Because C57BL6 mice have defective posterior communicating arteries, unilateral hyperintense DWI in this strain of mice is considered to arise from unilateral deficit in this communicating circulation. Investigating this link will be a focus of our future work. One representative mouse with unilateral hyperintense DWI was selected for illustration purposes, so that we could compare injured and less-injured hemispheres in the same mouse (Fig. 5
Ai).
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Again, to avoid possible complications of elevated GFAP expression during reactive gliosis and enhanced macrophages, which may adversely affect probe delivery immediately following acute neurological disorders, we delivered SPION-GRAP 8 wk after 60 min BCAO. DWIs obtained 1 day postischemia show metabolic disturbance in expanded areas of the striatum of all seven animals; we observed no obvious abnormal T2-weighted MR images at this time point, although we did observe ventriculomegaly in the hemisphere ipsilateral to hyperintense DWI (arrow in Fig. 5Av
). After delivering SPION-gfap in an eyedrop solution and allowing 1 day for uptake and distribution, we found not only hyperintense R2* maps in the brains of all animals with BCAO, but also foci of elevated retention in the ipsilateral striatum (arrow, Fig. 5Aii
), where BBB leakage was validated using Gd-DTPA 1 wk later (Fig. 5Aiii, iv
). We observed matching locations of hyperintense DWI, focal SPION retention, and BBB leakage in the ROI. However, neither ventriculomegaly nor hyperintense DWI was observed in any of the age- and sex-matched sham-operated animals (Fig. 5Bi
). We observed no anomalies in the whole-brain R2* maps after similar SPION-gfap application to these controls (Fig. 5Bii
). We obtained postmortem samples from four mice to validate gliosis by immunohistochemistry. As shown in Fig. 5Ci
, there was an absence of gliosis in the left striatum where we observed normal DWI (Fig. 5Ai
) and an absence of elevated SPION retention (Fig. 5Aii
). However, we did observe gliosis in the right striatum ipsilateral to the enlarged ventricle in the same mouse (Fig. 5Cii
). Matching patterns of gliosis were found in postmortem samples of all mice that showed hyperintense foci in R2* maps (n=4, 100%). The R2* values or retention of SPION in the brain were not statistically analyzed because each of the four animals with BCAO had different shapes and locations of hyperintense R2* foci. When the duration of cerebral ischemia was reduced from 60 to 30 min, we observed no hyperintense DWI, BBB leakage, or focal SPION retention after similar SPION-gfap delivery (n=4, 100%). In addition, the absence of an abnormal pattern of glia in sham-operated mice was validated in Fig. 5D
(MRI: n=4; histology, n=2). Glia in the striatum of sham-operated mice were noted as having a shorter axon (Fig. 5Di, ii
) than those in the same regions of BCAO-induced gliosis (Fig. 5Cii
, insets), suggesting few or no abnormal fibrous glial network in the sham-operated animals.
We used tMRI to demonstrate the specificity of SPION-sODN for differentiating endogenous mRNA in living brains. We serially delivered two different SPION-sODNS at different time points to the same animals that had been previously treated with BCAO and exhibited bilateral hyperintense DWI (n=4); we selected one representative mouse. Figure 6
Ai, ii shows uneven hyperintense DWI and ADC drop, that is, the injury is more severe in the right hemisphere than in the left. We acquired T2*-weighted images and selected animals with focal signal drop in the vicinity of severe injury (arrow, Fig. 6B
).
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To delineate the nature of this signal drop, we applied SPION-gfap and SPION-β-actin (4 mg Fe/kg, OTRD) in the 8th and 9th weeks, respectively. We acquired R2* maps 1 day after probe delivery. The subtraction maps showed specific focal retention of SPION-gfap in tissue below the right ventricle (arrowhead and open arrow, Fig. 6C
). In addition, we observed ventriculomegaly (Vm) and hippocampal atrophy (asterisks) in the right hemisphere (Fig. 6C, D
). Because the alignment of MR slices became less perfect as a result of cell death and brain tissue atrophy, cortical SPION retention near the surface coil was generally ignored in this study and is pending future investigation.
We compared patterns of SPION retention after SPION-β-actin delivery 1 wk later (Fig. 6D
), at which time we observed more hyperintense foci in R2* maps and SPION-β-actin focal patterns different and more widespread than those of SPION-gfap (short upward arrows in Figs. 6C, D
). The aggregate previously observed in the T2*-weighted MRI became hyperintense in the subtraction maps after SPION-β-actin (arrowheads, Fig. 6D
). One focal R2* value elevation (arrowhead, Fig. 6C, D
), we observed the intensity of elevation was higher in SPION-β-actin than those observed for SPION-gfap. We obtained postmortem samples in the 11th week to validate antigen expression in these foci by immunohistochemistry. The validation assay used antibodies against GFAP or β-actin (red), and counterstained for endothelia (green) in the blood vessels. We observed normal patterns of endothelia and glia in the left hemisphere, where we detected less hyperintense DWI (Fig. 6Ci
and inset). However, we observed intense gliosis (red) in the location where we also observed hyperintense R2* maps in the right hemisphere. The cells that expressed GFAP are not endothelia because GFAP-expressing cells did not merge with endothelia (inset in Fig. 6Cii-vi
). On the other hand, we observed groups of β-actin-expressing cells in the vicinity of R2* foci of SPION-β-actin (matching arrows in Fig. 6Di-vi
); the β-actin-expressing cells merged with the vascular endothelia (Fig. 6Dii, iii
).
We observed one common hyperintense aggregate in the thalamus and below the hippocampus, corresponding to a signal drop in the T2*-weighted MRI in Fig. 6B
and in all R2* maps of both SPION-gfap and SPION-β-actin (long arrows, Fig. 6C, D
). The intensity of this R2* lesion in the subtraction maps of SPION-β-actin is stronger than that of SPION-gfap (arrowheads, Fig. 6C, D
). The lesion appears to be an aggregate of endothelia containing β-actin antigen (Fig. 6Dii-vi
), though it may also contain a few GFAP-expressing cells (Fig. 6Cii
). We also observed that some endothelia did not express β-actin antigen; these cells were located at sites where we found no hyperintense R2*signal (asterisk, Fig. 6Div
). We conclude that our results demonstrate the specificity of SPION-sODN for reporting specialized cells with OTRD and tMRI in live animals.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 13)
. Now, we demonstrated here the same probe can report cells that express different levels of targeted mRNA. Moreover, different targeting probes are used in the same animal at different times for mRNA-based cell typing in MRI.
Here, we have demonstrated that our modular MR probe can be delivered to live brains noninvasively when the BBB is compromised. Puncture wounds and CSD are two examples of acute neurological disorders known to have characteristic BBB leakage. While puncture wound is representative of physical disruption of brain tissue, much like traumatic brain injury, the CSD model represents minimal physical injury with micro-BBB leakage and requires microscopic examination for Evans blue extravasation (22)
. Although BBB leakage after CSD may be short-lived (approximately 1 wk), such minor and transient disruption is sufficient to allow SPION retention and MR probe uptake in brain cells. On the other hand, the GCI model of BCAO is an acute neurological disorder thought to simulate the neurological conditions associated with cardiac arrest; it seldom induces necrosis but elicits oxidative DNA damage and apoptotic DNA fragmentation (27
28
29)
. Several reports have described how GCI induces BBB leakage in rat and mouse brains (30
; Savitz et al., unpublished data); we report here that BBB leakage in a C57BL6 mouse GCI model can be detected as early as 10 h and as late as 9 wk after BCAO when assessing Gd retention. BBB leakage allows detection of brain damage using specific MRI probes in live mice (8)
. When employing i.p. delivery of SPION-cfos after BCAO, R2* maps of elevated SPION retention after i.p. delivery are similar to maps acquired after i.c.v. delivery. We have obtained cortical R2* values (40 s–1) that represent levels somewhat lower than those measured within 1 day of cerebral ischemia with i.c.v. delivery (60 s–1) (8
, 13)
. We believe the difference may result from late administration of SPION-cfos probe in this model, when the expression of immediate early gene transcript was no longer at its peak (31)
. Expression eventually disappears altogether as a result of neuronal death (8)
. Our data support the notion that the sequence in the sODN of SPION-sODN dictates retention according to the intracellular level of the target mRNA (14)
. This notion is also demonstrated by the results obtained when using MR probes with different intracellular targets for cells that express GFAP and β-actin antigens.
Second, we have demonstrated that SPION-sODN probe complexes can be distributed in the brain when they are introduced to either the peritoneal cavity or the conjunctival sac. The results of our study suggest that the lymphatic and vascular circulation provide a connection between the peritoneal cavity or the conjunctival sac and the brain when the BBB is disrupted. However, the exact pathway is not yet fully understood. SPION-sODN can be distributed from the peritoneal cavity (by i.p. delivery) and through ocular fluid (via OTRD) to the CNS vasculature and can enter the brain cells via BBB leakage. Evidence of these capabilities of the SPION probe includes 1) the stronger R2* lesion hyperintensity in the subtraction maps of SPION-β-actin compared to SPION-gfap (arrows, Fig. 6C, D
), 2) the matching between the lesion and the signal reduction in T2*-weighted MRI (Fig. 6B
), 3) the aggregate of vascular tissue in the location of endothelia expressing β-actin antigen (Fig. 6Di-vii
), and 4) reporting of in vivo gliosis (Fig. 6Ciii
).
The prerequisite of BBB leakage for noninvasive delivery methods may indicate that OTRD or i.p. delivery has advantages over i.c.v. delivery in all applications, especially in human subjects who experience neurological disorders and BBB disruption. In some cases, patients who have special needs can be transiently treated with osmotic shock by mannitol to open BBB. OTRD is a user-friendly method, such that patients can self-administer the probe at home before reporting for the imaging examination. In addition, we can use repetitive applications of SPION-sODN with the same or different targets in the same subject, provided a clearance time is given to the subject of detection. Our current study indicates that tMRI and our MR probes are not limited to diagnosis of gliosis, angiogenesis, and brain repair, but also have potential targeting ability for glioma or cancer cells when mRNA of specific oncogenes are known. On the other hand, one disadvantage is that sealing of the BBB over time may either diminish or completely block MR probe distribution. Therefore, the assay used to determine BBB leakage is very important. We employed two conventional methods (Evans blue extravasation and tissue retention of Gd-DTPA). Whereas Evans blue extravasation terminates experiments altogether, Gd-DTPA retention is convenient and is washed clean the next day and as such can be used for predelivery testing for eligibility of tMRI using our unique MR probe.
We have demonstrated here that SPION-gfap and SPION-β-actin give different subtraction R2* maps. Because the sODN used in this study has been shown to support PCR or in situ hybridization of mRNA transcript (13
, 24
, 32)
, the basis for the specificity of these SPION-sODN probes resides in the complementarity between the sODN sequences and mRNA transcripts. Specific patterns of SPION retention, representing special mRNA transcript expression, are reflected by different cell types expressing specific mRNA of GFAP and β-actin antigens. For example, SPION-gfap detects in vivo gliosis where immunohistochemistry of postmortem samples also reveals a fibrous network of glia or astrocytes. The cell typing using SPION-sODN is based on the ability of our MRI probe to differentiate two levels of mRNA expression: the normal pattern of GFAP-expressing cells in Figs. 4E
and 5Ci
and the fibrous network of glia expressing elevated signal in Figs. 4D
and 5Cii
. Moreover, we employed subtraction R2* maps (Fig. 6)
to reduce background R2* attributable to endogenous retention of baseline glia. As such, subtraction R2*maps not only identify foci of gliosis but also report ROIs with abnormal expression of β-actin, resulting from some pathophysiological condition.
Such specificity of cell typing is also demonstrated for SPION-β-actin. Normally, β-actin mRNA is not elevated immediately after cerebral ischemia (32)
. Tissue remodeling that involves angiogenesis has been reported in stroke model of the rat using postmortem samples (33)
. Indeed, SPION-β-actin retention is elevated in areas close to regions where we identified ADC drops 1 day after GCI (Fig. 6Aii
). These regions were found to contain pericytes that express β-actin antigen in the vascular wall 9 wk after GCI. SPION-β-actin retention was not elevated in regions that exhibited gliosis, except in one aggregate of endothelia (arrows, Fig. 6B-D
). (At the 10th wk, we also tested the retention of SPION probe targeting matrix metalloproteinase-9 (MMP-9) mRNA, known to have the vascular origin during remodeling after stroke (33).) Although the retention of SPION-mmp9 matches those of SPION-βactin, the assay is pending for suitable polyclonal antibodies for mouse MMP-9 antigen.
Therefore, our results agree with other reports showing that β-actin-expressing cells can be detected during scarring (34)
and in CNS microvascular pericytes during angiogenesis (35
, 36)
. Future investigations will be needed to validate this reporting capability of SPION-β-actin for angiogenesis.
We have developed, validated, and applied in vivo techniques using tMRI. We have demonstrated transcript imaging of glia and cells expressing smooth muscle actin in new endothelia during the repair process. Brain repair after neurological disorders has an important role in plasticity (37)
. This tMRI technology thus represents a breakthrough for molecular biologists of diverse disciplines, and the availability of this method and contrast probe that targets endogenous gene activities will enable real-time investigation and longitudinal tracking of pathophysiological conditions in living systems. Noninvasive methods of delivering modular probes to the brain also lend themselves to other clinical applications such as noninvasive targeting of gene action in cells when certain mRNA transcripts are known to impact a specific pathological change.
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ACKNOWLEDGMENTS |
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Note added in proof: We now have performed BBB leakage determination in mice using Evans blue extravasation (n=8) or Gd-MRI at 1 (n=6), 7 (n=10), and 24 (n=2) h after GCI of 60 min. We found BBB leakage in 4 mice, or 22%, within the first day of GCI. All mice that showed hyperintense DWI (with significant ADC drop) 1 day after GCI in the striatum developed striatal BBB leakage.
Received for publication August 27, 2007. Accepted for publication October 18, 2007.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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