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Experimental Studies |
1 From the Departments of Obstetrics and Gynecology (N.M., T. Kubota, T.A.), Neuropathology (T. Kuroiwa), Neurology (F.Y.Z.), Neurosurgery (T.N., H.A.), and Radiology (I.Y.), Faculty of Medicine, Tokyo Medical and Dental University, Yushima 1-5-45, Bunkyo-ku, Tokyo 113-8519 Japan. Received February 26, 1999; revision requested May 6; revision received August 9; accepted August 18. Address reprint requests to N.M. (e-mail: n.miyasaka.gyne@med.tmd.ac.jp).
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Abstract |
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 TOP Abstract IntroductionMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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MATERIALS AND METHODS: Fifteen 3-week-old rats were subjected to a 30-minute IH insult (unilateral common carotid arterial ligation and exposure to 8% oxygen) and were examined at diffusion-weighted magnetic resonance imaging and light and electron microscopy on cessation of the insult (n = 5), 60 minutes after resuscitation (n = 5), or 48 hours after resuscitation (n = 5). Twelve control rats either underwent unilateral common carotid arterial ligation or were subjected to hypoxia.
RESULTS: The experimental rats showed primary ADC reduction during the insult, transient ADC recovery after resuscitation, and secondary ADC reduction 48 hours after the insult. Histologic examination revealed dendritic swelling and mild swelling of the perivascular astrocytic end-feet during the primary ADC reduction phase, dark neurons and pronounced swelling of the perivascular astrocytic end-feet during the transient ADC recovery phase, and severely retracted dark neurons and extensive swelling of the astrocytic end-feet during the secondary ADC reduction phase.
CONCLUSION: Transient ADC normalization after cerebral IH does not necessarily mean that histologic normalization has occurred. The transient ADC recovery phase appeared to have limited potential for neuronal salvage.
Index terms: Animals • Brain, ischemia, 10.781 • Brain, MR, 10.121411, 10.12144, 10.781 • Magnetic resonance (MR), comparative studies • Magnetic resonance (MR), diffusion study, 10.121411, 10.12144 • Magnetic resonance (MR), experimental studies
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Introduction |
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TOPAbstractIntroductionMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Moreover, investigators in a recent study of human stroke (4) found pseudonormalized ADCs in regions of cerebral infarction within 10 hours of onset. However, the meaning of ADC normalization is still not clear, and whether the transient ADC normalization after cerebral IH indicates histologic normalization is not clarified. In this study, we examined the ADC changes and histologic findings in young rats with cerebral IH at three different stages—primary ADC reduction, transient ADC recovery, and secondary ADC reduction. We evaluated the relationship between the temporal changes in the ADC and the histologic findings to assess whether there is a potential therapeutic window after cerebral IH.
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MATERIALS AND METHODS |
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TOPAbstractIntroductionMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Experimental Protocol
The 27 rats were assigned to three groups: Group A (n = 6) underwent unilateral common carotid arterial ligation alone, group B (n = 6) was subjected to hypoxia alone, and group C (n = 15) underwent unilateral common carotid arterial ligation and was subjected to hypoxia. Baseline diffusion-weighted MR imaging was performed. Afterwards, the rats in groups B and C were subjected to hypoxia caused by reducing the inspired oxygen concentration to 8% by means of dilution with nitrogen. Diffusion-weighted MR imaging was repeated during the hypoxic state, and ADC changes in the ipsilateral cerebral cortex were monitored by using ADC maps that were generated as soon as data acquisition was complete.
Histologic examination of five of the 15 rats in group C (group C-1) was performed 30 minutes after the onset of ADC reduction. The other 10 rats were resuscitated by increasing the oxygen concentration to 30% 30 minutes after the onset of ADC reduction, and diffusion-weighted MR imaging was repeated 60 minutes after the start of resuscitation. Then, histologic examination of five of these 10 rats (group C-2) was performed, and the other five (group C-3) were returned to their cages after they awoke from the anesthesia. Diffusion-weighted MR imaging in group C-3 was performed 48 hours after the IH insult and was followed by histologic examination.
The rats in group B (n = 6) were resuscitated 30 minutes after the onset of hypoxia. They were imaged according to the previous protocol, and histologic examination was performed during the hypoxic state (n = 2), 60 minutes after resuscitation (n = 2), or 48 hours after resuscitation (n = 2). The rats in group A (n = 6) were imaged and underwent histologic examination 1 (n = 2), 2 (n = 2), or 48 (n = 2) hours after unilateral common carotid arterial ligation.
MR Imaging Examination
MR imaging was performed by using a 4.7-T superconducting system with a 23-cm horizontal-bore magnet and a 65 mT/m maximum gradient capability (Unity INOVA; Varian, Palo Alto, Calif). A quadrature coil with an internal diameter of 8 cm was tuned to 200 MHz for radio-frequency excitation and MR signal reception. After the surgical procedure, each rat was placed in a supine position on a thermal water blanket maintained at 37.5°C, was artificially ventilated with 1.5% isoflurane in a mixture of 70% nitrous oxide and 30% oxygen, was immobilized with intravenous injections of pancuronium bromide (Mioblock; Organon Teknika, Boxtel, the Netherlands; 100 mg per kilogram of body weight), and was placed in an MR imaging–compatible stereotactic frame to prevent motion artifacts. The inspired oxygen, or FIO2, and expiratory carbon dioxide, or PCO2, concentrations were continuously monitored throughout the experiment.
Diffusion-weighted MR imaging was performed with multisection spin-echo sequences with a repetition time msec/echo time msec of 1,500/80, a matrix of 128 x 64, a field of view of 30 x 30 mm, a section thickness of 2 mm without an intersection gap, and one signal acquired. The diffusion gradients (duration, 30.5 msec; separation time, 36.7 msec; gradient strength, 0 or 26 mT/m) were applied along the three orthogonal directions (x, y, and z axes). The resultant value for the gradient factor b was 0 or 1,200 sec/mm2. Diffusion-weighted MR imaging with a b value of 0 sec/mm2 was used at T2-weighted MR imaging. The acquisition time for one set of diffusion-weighted images was 6.4 minutes.
Data Analysis
All analyses were performed by using a Sun Sparc 10 workstation (Sun Microsystems, Mountain View, Calif) and image analyzing software (XDS software, Davis Bioengineering, St Louis, MO). ADC maps were plotted on a pixel-by-pixel basis by using the following equation (5): ADC = ln(S0/S1)/(b1 - b0), where S0 and S1 are the signal intensities of the two diffusion-weighted images that represent the mean of three orthogonal planes (ie, a trace of the diffusion tensor), and b0 and b1 are 0 and 1,200 sec/mm2, respectively. A section (1.8 mm posterior to the bregma) was chosen, and regions of interest for the ADC measurements were drawn on the ipsilateral parietal cortex (Fig 1). The regions of interest were drawn by one author (T.N.), and the ADC measurements were obtained by another author (N.M.)
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Statistical Analysis
Between-group data were compared by performing an unpaired Student t test, and the differences from the baseline values of each group were analyzed by using a paired t test. Differences with P values of less than .05 were considered significant.
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RESULTS |
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TOPAbstractIntroductionMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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The mean ADC values in the ipsilateral parietal cortices of these experimental rats were (7.04 ± 0.59) x 10-4 mm2/sec before the IH insult, (4.58 ± 0.63) x 10-4 mm2/sec on cessation of the IH insult, (7.00 ± 0.31) x 10-4 mm2/sec 60 minutes after resuscitation, and (4.78 ± 0.38) x 10-4 mm2/sec 48 hours after the IH insult (Fig 1b). ADC values on cessation of and 48 hours after IH insult were significantly lower than those before IH insult (both P <.05). However, those measured 60 minutes after resuscitation and before IH insult did not differ significantly, which indicated that transient ADC normalization had occurred.
Comparison of ADC Changes and Histologic Findings
Figure 2 shows T2-weighted MR images, ADC maps, and photomicrographs of control and experimental rats. Figure 3 shows the corresponding electron microscopic findings. In the control rats, neurons were normal and cell density in the ipsilateral parietal cortex was well preserved at all time points (Figs 2a, 3a).
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T2-weighted MR images obtained during the transient ADC recovery phase also showed normal signal intensity in the affected regions. Light microscopy revealed that neuropilar microvacuolation and perivascular space enlargement were more severe in this phase than during the primary ADC reduction phase, and many neurons (approximately 50–70 neurons per high-power filed) had pyknotic nuclei (Fig 2c). Electron microscopy revealed that swelling of the perivascular astrocytic end-feet was more pronounced in this phase than during the primary ADC reduction phase (Fig 3c). Furthermore, the perineural astrocytic processes were swollen, and many neurons showed cell body retraction with increased electron density and nuclear pyknosis (dark neuronal changes). The cytoplasmic membranes of these neurons were disrupted (Fig 3c).
T2-weighted images obtained during the secondary ADC reduction phase showed high signal intensity in the affected regions. Light microscopy revealed that neuropilar microvacuolation and perivascular space enlargement were far more severe in this phase than during the transient ADC recovery phase, and all neurons had pyknotic nuclei (Fig 2d). Electron microscopy revealed severely swollen perivascular astrocytic end-feet (Fig 3d) and severely retracted dark neurons surrounded by swollen astrocytic processes. However, all limiting cell membranes of the swollen astrocytic processes appeared to be intact (Fig 3d).
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DISCUSSION |
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TOPAbstractIntroductionMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Primary ADC reduction was observed in the cerebral hemisphere ipsilateral to the side of common carotid arterial ligation during IH insult. Acute ADC reductions during experimental and human cerebral ischemia have been described (6,7) and were demonstrated to delineate ischemic cerebral changes as early as 5–10 minutes after the onset of ischemia (8,9).
Although the mechanisms responsible for ADC reduction are not completely understood, the extracellular space has been considered to be the main determinant of the effective ADC (6,10,11). The extracellular spaces in ischemic tissue become smaller as water shifts into the intracellular space as a result of increased intracellular osmolarity (12) and impairment of ion pumps in the cell membrane.
Our histologic examinations performed during this phase revealed dendritic swelling, which is one of the earliest changes caused by IH cerebral damage (13,14). We also observed swelling of the perivascular astrocytic end-feet, which, in our previous study (15), occurred during the early phase of focal cerebral ischemia in cats. Both of these findings correlated well with the ADC reduction observed during the IH insult. The neurons with pale nuclei and swollen cytoplasm observed during this phase are probably compatible with the morphologic changes previously referred to as reactive changes (16); this finding indicates that the histologic changes are potentially reversible.
Our data demonstrated that resuscitation after IH insult led to complete normalization of the ADC in the cerebral cortex; this finding was consistent with that of a previous study in which an adult rat model of cerebral IH was used (3). However, in contrast to this ADC normalization, our histologic examination revealed pronounced hydropic swelling of the perivascular astrocytic end-feet and many dark neurons in all affected regions. To our knowledge, no report of the histologic findings observed during the ADC recovery phase after IH insult has been published.
Paradoxical normalization of ADC that occurs days after an ischemic event is a well-known clinical observation; it has been attributed to a balance of cytotoxic and vasogenic edema. However, the transient ADC normalization observed in this study appeared to be another case because it was not associated with areas of high signal intensity on T2-weighted MR images. Our results demonstrated the simultaneous presence of astrocytic swelling and neuronal cell retraction during this phase; these histologic findings appeared to contribute to the transient ADC normalization. Astrocytic swelling could reduce the ADC by reducing the extracellular space, whereas neuronal cell retraction with cell membrane disruption could elevate the ADC by increasing the extracellular space. Consequently, these two events in combination could normalize the ADC during this phase. In this regard, other mechanisms such as changes in the permeability of the cell membranes to water (17) or changes in the diffusion of intracellular water (18,19) should also be taken into account in the explanation of this ADC normalization.
Whatever the mechanisms responsible for ADC normalization are, our data clearly demonstrate that transient ADC recovery after resuscitation from IH insult does not necessarily mean that histologic normalization has occurred. This finding is important because ADC recovery after reperfusion in models of focal cerebral ischemia has been considered to reflect tissue salvage (20–22). As discussed previously, the transient ADC recovery phase after cerebral IH has also been considered to be a potential therapeutic window (3). However, the dark neuronal changes with membrane disruption that we observed in most neurons during this phase indicated that the neuronal damage was irreversible. Thus, our data indicate that the transient ADC recovery phase may have limited potential for neuronal salvage, although it still has potential as a therapeutic window to prevent further progression of cerebral infarction.
Secondary ADC reduction occurred 48 hours after IH insult and was accompanied by areas of very high signal intensity on T2-weighted MR images. These findings differed from those associated with focal cerebral ischemia at the chronic stage during which both ADC elevation and T2 elongation have been reported (23). In our study, electron microscopy revealed swelling of the perivascular astrocytic end-feet with intact cell membranes, whereas cell membrane fragmentation during the chronic stage of focal cerebral ischemia has been reported (24). Furthermore, although the simultaneous presence of astrocytic swelling and neuronal cell retraction during this phase was similar to that observed during the transient ADC recovery phase, light microscopy revealed that the astrocytic swelling was far more severe during the secondary ADC reduction phase (Fig 2). Thus, the presence of severely swollen astrocytes with intact cell membranes appeared to correspond to secondary ADC reduction.
The evaluation of cerebral perfusion is important in the characterization of ischemic cerebral damage, but we did not perform dynamic T2*-weighted MR imaging to evaluate the time course of perfusion changes. In a rat model of cerebral IH (unilateral common carotid arterial occlusion and subsequent hypoxia), systemic blood pressure is known to decrease during hypoxia; this decrease induces cerebral hypoperfusion or ischemia. Cerebral blood flow in the contralateral cerebral hemisphere also changes during hypoxia and after resuscitation (3). Therefore, cerebral perfusion parameters measured with dynamic T2*-weighted MR imaging no longer provide quantitative information about the changes in cerebral perfusion. Furthermore, our objective in this study was not to examine the correlation between the changes in cerebral perfusion and ADC but to investigate the relationship between ADC and histologic changes. Thus, we did not perform T2*-weighted MR imaging in this study.
Finally, although we intended to examine the radiologic-histologic correlates of cerebral IH in neonates, findings from recent studies of experimental focal cerebral ischemia have demonstrated the delayed (secondary) ADC decrease that occurs several hours to days after reperfusion (24). We believe that a study of the time course of the histologic changes should be undertaken to clarify the meaning of ADC normalization after reperfusion. The findings will be of value in the treatment of patients with ischemic stroke.
Practical application: Diffusion-weighted MR imaging is a powerful technique that can demonstrate IH cerebral damage at a very early stage after the causative insult. Although this technique has been and will be used for the clinical evaluation of human neonates who experienced asphyxiation at birth, the transient ADC normalization after cerebral IH does not necessarily mean that histologic normalization has occurred. Despite normalization of the ADC, progressive, irreversible, histologic neuronal changes occur after cerebral IH.
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Acknowledgments |
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Footnotes |
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Author contributions: Guarantor of integrity of entire study, N.M.; study concepts, N.M., T.A.; study design, N.M., T. Kuroiwa; definition of intellectual content, T. Kuroiwa, T.N.; literature research, N.M., T.N.; experimental studies, N.M., T. Kuroiwa, F.Y.Z.; data acquisition, N.M., F.Y.Z.; data analysis, T. Kuroiwa, N.M.; statistical analysis, H.A.; manuscript preparation, N.M.; manuscript editing, I.Y., T. Kubota; manuscript review, T. Kuroiwa, T.A.
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References |
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TOPAbstractIntroductionMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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