Wolfram Schwindt

Monitoring of implanted stem cell migration in vivo: A highly resolved in vivo magnetic resonance imaging investigation of experimental stroke in rat

In vivo monitoring of stem cells after grafting is essential for a better understanding of their migrational dynamics and differentiation processes and of their regeneration potential. Migration of endogenous or grafted stem cells and neurons has been described in vertebrate brain, both under normal conditions from the subventricular zone along the rostral migratory stream and under pathophysiological conditions, such as degeneration or focal cerebral ischemia. Those studies, however, relied on invasive analysis of brain sections in combination with appropriate staining techniques. Here, we demonstrate the observation of cell migration under in vivo conditions, allowing the monitoring of the cell dynamics within individual animals, and for a prolonged time. Embryonic stem (ES) cells, constitutively expressing the GFP, were labeled by a lipofection procedure with a MRI contrast agent and implanted into rat brains. Focal cerebral ischemia had been induced 2 weeks before implantation of ES cells into the healthy, contralateral hemisphere. MRI at 78-μm isotropic spatial resolution permitted the observation of the implanted cells with high contrast against the host tissue, and was confirmed by GFP registration. During 3 weeks, cells migrated along the corpus callosum to the ventricular walls, and massively populated the borderzone of the damaged brain tissue on the hemisphere opposite to the implantation sites. Our results indicate that ES cells have high migrational dynamics, targeted to the cerebral lesion area. The imaging approach is ideally suited for the noninvasive observation of cell migration, engraftment, and morphological differentiation at high spatial and temporal resolution.

Differential Effects of NMDA and AMPA Glutamate Receptors on Functional Magnetic Resonance Imaging Signals and Evoked Neuronal Activity during Forepaw Stimulation of the Rat

Most of the currently used methods for functional brain imaging do not visualize neuronal activity directly but rather rely on the elicited hemodynamic and/or metabolic responses. Glutamate, the major excitatory neurotransmitter, plays an important role in the neurovascular/neurometabolic coupling, but the specific mechanisms are still poorly understood. To investigate the role of the two major ionotropic glutamate receptors [NMDA receptors (NMDA-Rs) and AMPA receptors (AMPA-Rs)] for the generation of functional magnetic resonance imaging (fMRI) signals, we used fMRI [measurements of blood oxygenation level-dependent (BOLD), perfusion-weighted imaging (PWI), and cerebral blood volume (CBV)] together with recordings of somatosensory evoked potentials (SEPs) during electrical forepaw stimulation in the α-chloralose anesthetized rat. Intravenous injection of the NMDA-R antagonist MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d] cyclohepten-5,10-imine maleate] (0.06 mg/kg plus 3.6 μg · kg−1 · h−1) significantly decreased BOLD (−51 ± 19%; n = 5) and PWI (−57 ± 26%; n = 5) responses but reduced the SEPs only mildly (approximately −10%). Systemic application of the AMPA-R antagonist GYKI-53655 [1-(4-aminophenyl)-3-methylcarbamyl-4-methyl7,8-methylenedioxy-3,4-dihydro-5H-2,3-benzodiazepine] significantly decreased both the hemodynamic response (BOLD, −49 ± 13 and −65 ± 15%; PWI, −22 ± 48 and −68 ± 4% for 5 and 7 mg/kg, i.v., respectively; CBV, −80 ± 7% for 7 mg/kg; n = 4) and the SEPs (up to −60%). These data indicate that the interaction of glutamate with its postsynaptic and/or glial receptors is necessary for the generation of blood flow and BOLD responses and illustrate the differential role of NMDA-Rs and AMPA-Rs in the signaling chain leading from increased neuronal activity to the hemodynamic response in the somatosensory cortex.

Functional uncoupling of hemodynamic from neuronal response by inhibition of neuronal nitric oxide synthase.

The cerebrovascular coupling under neuronal nitric oxide synthase (nNOS) inhibition was investigated in α-chloralose anesthetized rats. Cerebral blood flow (CBF), cerebral blood volume (CBV), and blood oxygenation level dependent (BOLD) responses to electrical stimulation of the forepaw were measured before and after an intraperitoneal bolus of 7-nitroindazole (7-NI), an in vivo inhibitor of the neuronal isoform of nitric oxide synthase. Neuronal activity was measured by recording somatosensory-evoked potentials (SEPs) via intracranial electrodes. 7-Nitroindazole produced a significant attenuation of the activation-elicited CBF (P < 10−6), CBV (P < 10−6), and BOLD responses (P < 10−6), without affecting the baseline perfusion level. The average ΔCBF was nulled, while ΔBOLD and ΔCBV decreased to ~30% of their respective amplitudes before 7-NI administration. The average SEP amplitude decreased (P < 10−5) to ~60% of its pretreatment value. These data describe a pharmacologically induced uncoupling between neuronal and hemodynamic responses to functional activation, and provide further support for the critical role of neuronally produced NO in the cerebrovascular coupling.

Dynamic changes of ADC, perfusion, and NMR relaxation parameters in transient focal ischemia of rat brain

The potential of multiparametric MRI parameters for differentiating between reversibly and irreversibly damaged brain tissue was investigated in an experimental model of focal brain ischemia in the rat. The middle cerebral artery (MCA) was occluded by intraluminal suture insertion for 60 or 90 min, followed by 4.5 h of reperfusion. The apparent diffusion coefficient (ADC) of brain water, T1 and T2 relaxation times, and CBFi, an MR‐derived index of cerebral perfusion, were repeatedly measured and correlated with the outcome from the ischemic impact. A novel user‐independent approach for segmentation of ADC maps into classes of increasing injury was introduced to define regions of interest (ROIs) in which these parameters were evaluated. MCA occlusion led to a graded decline of ADC, which corresponded with both the severity of flow reduction and an increase in T1 and T2 relaxation times. Removal of the suture led to a triphasic restitution of blood flow consisting of a fast initial rise, a secondary decline, and final normalization. Postischemic reperfusion led to a rise of ADC irrespective of the duration of ischemia. However, the quality of recovery declined with increasing severity of the ischemic impact. Throughout the observation time, T1 and T2 showed a continuous increase, the intensity of which correlated with the severity of ADC decline during ischemia. Particularly with longer ischemia time, elevated T2 in combination with reduced ADC yielded a lower probability of recovery during recirculation, while intraischemic perfusion information contributed less to the prediction of outcome. In conclusion, the combination of MR parameters at the end of ischemia correlated with the probability of tissue recovery but did not permit reliable differentiation between reversibly and irreversibly damaged tissue. Magn Reson Med 47:97–104, 2002.

Gradient echo time dependence and quantitative parameter maps for somatosensory activation in rats at 7 T

The dependence of functional magnetic resonance imaging (MRI) contrast on the gradient echo time TE in T*2‐weighted blood oxygenation level‐dependent (BOLD) fast low‐angle shot (FLASH) imaging has been studied at 7 T for electrical forepaw stimulation in α‐chloralose anesthetized rats. The observed variation of both the activation signal intensity and spatial pattern with echo time TE, resulting from the regional heterogeneity of T*2, was assessed by the calculation of quantitative T*2 and quantitative STE=0 maps, the latter representing the back‐extrapolated signal intensity for TE = 0. The subsequently determined T*2 and STE=0 activation maps allowed a pixelwise separation of true BOLD from inflow contributions to forepaw stimulation‐induced signal change in the somatosensory cortex of rat brain. For functional activation experiments performed with one single echo time the prior measurement of a quantitative T*2 map is recommended as minimum further information to judge the intensity and the regional pattern of the resulting activation maps. Magn Reson Med 42:118–126, 1999.

Facilitation of electric forepaw stimulation‐induced somatosensory activation in rats by additional acoustic stimulation: An fMRI investigation

The influence of scanner acoustic noise on somatosensory activation pattern in rat cortex was investigated by functional magnetic resonance imaging (fMRI) using the blood oxygenation level‐dependent (BOLD) contrast. This was achieved by two approaches. The first approach was to compare a conventional, loud fMRI sequence with a new sequence, in which the noise level was reduced by about 30 dB. In the second approach, the inner ear of the animal was destroyed, resulting in deafness. We compared the activation patterns obtained with both sequences before and after cochleotomy. The activated area was larger when data were sampled with background noise, and was also larger before cochleotomy than after. Thus, facilitation of somatosensory activation is induced by additional acoustic stimulation. Magn Reson Med 44:317–321, 2000.

Arterial spin tagging perfusion imaging of rat brain dependency on magnetic field strength

Perfusion-weighted imaging (PWI), using the method of arterial spin tagging, is strongly T(1)-dependent. This translates into a high field dependency of the perfusion signal intensity. In order to determine the expected signal improvement at higher magnetic fields we compared perfusion-weighted images in rat brain at 4.7 T and 7 T. Application of PWI to focal ischemia and functional activation of the brain and the use of two different anesthetics allowed the observation of a wide range of flow values. For all these (patho-)physiological conditions switching from 4.7 T to 7 T resulted in a significant increase of mean perfusion signal intensity by a factor of 2.96. The ratio of signal intensities of homotopic regions in the ipsi- and contralateral hemisphere was field-independent. The relative contribution of a) T(1) relaxation time, b) net magnetization, c) the Q-value of the receiver coils and d) the degree of adiabatic inversion to the signal improvement at higher field strength were discussed. It was shown that the main parameters contributing to the higher signal intensity are the lengthening of T(1) and the higher magnetization at the higher magnetic field.

Computed Tomographic Blend Sign Is Associated With Computed Tomographic Angiography Spot Sign and Predicts Secondary Neurological Deterioration After Intracerebral Hemorrhage

Background and Purpose— Significant early hematoma growth in patients with intracerebral hemorrhage is an independent predictor of poor functional outcome. Recently, the novel blend sign (BS) has been introduced as a new imaging sign for predicting hematoma growth in noncontrast computed tomography. Another parameter predicting increasing hematoma size is the well-established spot sign (SS) visible in computed tomographic angiography. We, therefore, aimed to clarify the association between established SS and novel BS and their values predicting a secondary neurological deterioration. Methods— Retrospective study inclusion criteria were (1) spontaneous intracerebral hemorrhage confirmed on noncontrast computed tomography and (2) noncontrast computed tomography and computed tomographic angiography performed on admission within 6 hours after onset of symptoms. We defined a binary outcome (secondary neurological deterioration versus no secondary deterioration). As secondary neurological deterioration, we defined (1) early hemicraniectomy under standardized criteria or (2) secondary decrease of Glasgow Coma Scale of >3 points, both within the first 48 hours after symptom onset. Results— Of 182 patients with spontaneous intracerebral hemorrhage, 37 (20.3%) presented with BS and 39 (21.4%) with SS. Of the 81 patients with secondary deterioration, 31 (38.3%) had BS and SS on admission. Multivariable logistic regression analysis identified hematoma volume (odds ratio, 1.07 per mL; P⩽0.001), intraventricular hemorrhage (odds ratio, 3.08; P=0.008), and the presence of BS (odds ratio, 11.47; P⩽0.001) as independent predictors of neurological deterioration. Conclusions— The BS, which is obtainable in noncontrast computed tomography, shows a high correlation with the computed tomographic angiography SS and is a reliable predictor of secondary neurological deterioration after spontaneous intracerebral hemorrhage.

CO2 Reactivity Measured by Perfusion MRI During Transient Focal Cerebral Ischemia in Rats

Background and Purpose CO2 response was examined in rats undergoing 60 minutes of middle cerebral artery occlusion (MCAO) and 4.5 hours of reperfusion. Because it is not clear whether the vasoreactivity improves during reperfusion in parallel with tissue recovery, CO2 response was determined spatially resolved, sequentially in the initially ischemic but later recovered areas and in the permanently damaged areas. Methods Apparent diffusion coefficient (ADC) maps were calculated from diffusion-weighted images, whereas CO2 reactivity maps were determined from the difference in perfusion signal intensity before and after CO2 stimulation. CO2 reactivity (administration of 6% CO2 for 5 minutes) was expressed in % change of perfusion signal intensity/mm Hg of Pco2 increase. ATP levels of tissue were used as a measure of outcome. The recovered and permanently damaged tissues were differentiated by combined use of end-ischemic ADC map and ATP image at the end of the experiment. Results The preischemic (control) CO2 reactivity of 3.5±0.9%/mm Hg decreased dramatically during MCAO in the ischemic hemisphere. During reperfusion, it remained <1%/mm Hg in the region with end-ischemic ADC <80% of the preischemic control value, but showed gradual recovery in the region with end-ischemic ADC >80% of control. Although at the end of the experiment the CO2 reactivity was significantly higher in the recovered tissue than in the permanently damaged tissue (1.15±0.44 and 0.13±0.47%/mm Hg, respectively;P <0.01), it still remained far below the normal control value (P <0.01). Conclusions The noninvasive perfusion-weighted MR imaging in combination with a CO2 challenge permits the investigation of the spatially resolved vascular reactivity during a longitudinal study of cerebral ischemia. Our data suggest that severe ischemia is followed by a prolonged disturbance of CO2 reactivity, despite already normalized energy metabolism.

Functional magnetic resonance imaging and somatosensory evoked potentials in rats with a neonatally induced freeze lesion of the somatosensory cortex

Brain plasticity is an important mechanism for functional recovery from a cerebral lesion. The authors aimed to visualize plasticity in adult rats with a neonatal freeze lesion in the somatosensory cortex using functional magnetic resonance imaging (fMRI), and hypothesized activation outside the primary projection area. A freeze lesion was induced in the right somatosensory cortex of newborn Wistar rats (n = 12). Sham-operated animals (n = 7) served as controls. After 6 or 7 months, a neurologic examination was followed by recording of somatosensory evoked potentials (SSEPs) and magnetic resonance experiments (anatomical images, fMRI with blood oxygen level–dependent contrast and perfusion-weighted imaging) with electrical forepaw stimulation under α-chloralose anesthesia. Lesioned animals had no obvious neurologic deficits. Anatomical magnetic resonance images showed a malformed cortex or hyperintense areas (cysts) in the lesioned hemisphere. SSEPs were distorted and smaller in amplitude, and fMRI activation was significantly weaker in the lesioned hemisphere. Only in a few animals were cortical areas outside the primary sensory cortex activated. The results are discussed in respect to an apparent absence of plasticity, loss of excitable tissue, the excitability of the lesioned hemisphere, altered connectivity, and a disturbed coupling of increased neuronal activity to the hemodynamic response.