Overexpression of adenosine kinase in cortical astrocytes and focal neocortical epilepsy in mice

Laboratory investigation

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Object

New experimental models and diagnostic methods are needed to better understand the pathophysiology of focal neocortical epilepsies in a search for improved epilepsy treatment options. The authors hypothesized that a focal disruption of adenosine homeostasis in the neocortex might be sufficient to trigger electrographic seizures. They further hypothesized that a focal disruption of adenosine homeostasis might affect microcirculation and thus offer a diagnostic opportunity for the detection of a seizure focus located in the neocortex.

Methods

Focal disruption of adenosine homeostasis was achieved by injecting an adeno-associated virus (AAV) engineered to overexpress adenosine kinase (ADK), the major metabolic clearance enzyme for the brain's endogenous anticonvulsant adenosine, into the neocortex of mice. Eight weeks following virus injection, the affected brain area was imaged via optical microangiography (OMAG) to detect changes in microcirculation. After completion of imaging, cortical electroencephalography (EEG) recordings were obtained from the imaged brain area.

Results

Viral expression of the Adk cDNA in astrocytes generated a focal area (~ 2 mm in diameter) of ADK overexpression within the neocortex. OMAG scanning revealed a reduction in vessel density within the affected brain area of approximately 23% and 29% compared with control animals and the contralateral hemisphere, respectively. EEG recordings revealed electrographic seizures within the focal area of ADK overexpression at a rate of 1.3 ± 0.2 seizures per hour (mean ± SEM).

Conclusions

The findings of this study suggest that focal adenosine deficiency is sufficient to generate a neocortical focus of hyperexcitability, which is also characterized by reduced vessel density. The authors conclude that their model constitutes a useful tool to study neocortical epilepsies and that OMAG constitutes a noninvasive diagnostic tool for the imaging of seizure foci with disrupted adenosine homeostasis.

Abbreviations used in this paper:AAV = adeno-associated virus; ADK = adenosine kinase; AP = anteroposterior; CBF = cerebral blood flow; DV = dorsoventral; EEG = electroencephalography; GFAP = glial fibrillary acidic protein; ML = mediolateral; NCE = neocortical epilepsy; OMAG = optical microangiography; SEM = standard error of the mean; TLE = temporal lobe epilepsy.

Abstract

Object

New experimental models and diagnostic methods are needed to better understand the pathophysiology of focal neocortical epilepsies in a search for improved epilepsy treatment options. The authors hypothesized that a focal disruption of adenosine homeostasis in the neocortex might be sufficient to trigger electrographic seizures. They further hypothesized that a focal disruption of adenosine homeostasis might affect microcirculation and thus offer a diagnostic opportunity for the detection of a seizure focus located in the neocortex.

Methods

Focal disruption of adenosine homeostasis was achieved by injecting an adeno-associated virus (AAV) engineered to overexpress adenosine kinase (ADK), the major metabolic clearance enzyme for the brain's endogenous anticonvulsant adenosine, into the neocortex of mice. Eight weeks following virus injection, the affected brain area was imaged via optical microangiography (OMAG) to detect changes in microcirculation. After completion of imaging, cortical electroencephalography (EEG) recordings were obtained from the imaged brain area.

Results

Viral expression of the Adk cDNA in astrocytes generated a focal area (~ 2 mm in diameter) of ADK overexpression within the neocortex. OMAG scanning revealed a reduction in vessel density within the affected brain area of approximately 23% and 29% compared with control animals and the contralateral hemisphere, respectively. EEG recordings revealed electrographic seizures within the focal area of ADK overexpression at a rate of 1.3 ± 0.2 seizures per hour (mean ± SEM).

Conclusions

The findings of this study suggest that focal adenosine deficiency is sufficient to generate a neocortical focus of hyperexcitability, which is also characterized by reduced vessel density. The authors conclude that their model constitutes a useful tool to study neocortical epilepsies and that OMAG constitutes a noninvasive diagnostic tool for the imaging of seizure foci with disrupted adenosine homeostasis.

Disruption of adenosine homeostasis is a pathological hallmark of temporal lobe epilepsy (TLE), and therapeutic adenosine augmentation is a rational approach for seizure control.5–7 Adenosine homeostasis in the brain is largely under the control of adenosine kinase (ADK), the key metabolic clearance enzyme for adenosine.2 In the adult brain ADK is predominantly expressed in astrocytes,59 where the expression levels of the enzyme determine the extent of a transmembrane gradient for adenosine, which, under baseline conditions, drives the influx of adenosine into the astrocyte through equilibrative nucleoside transporters.7,9,15,19 Therefore, synaptic levels of adenosine are largely under the control of astrocytes, which form a sink for the metabolic clearance of adenosine.9,15,17 We have previously demonstrated that astrogliosis and associated pathological overexpression of ADK is linked to neuronal hyperexcitability and seizure activity in rodent models of TLE,3,40 whereas surgically resected specimens from human patients with TLE were likewise characterized by profound overexpression of ADK.3,43 Conversely, genetic or virus-induced reduction of ADK expression in the hippocampus was shown to suppress seizures,40,60 while focal cell-based adenosine augmentation to the hippocampus was shown to protect the hippocampal formation from injury and seizures.40,41 Together, these findings demonstrate that adenosine homeostasis, controlled by astrocytic ADK, critically determines excitability of the hippocampus. However, whether this is true for other brain regions and other forms of epilepsy has not been investigated.

In addition to its direct control of neuronal function, adenosine signaling regulates vascular functions, which may contribute to the brain's susceptibility to seizures.1,47,50,51 Acutely, adenosine promotes hemodynamic events, such as vasodilation to increase blood flow in the brain, in response to hypoxia or ischemia.12,50,51 Adenosine, largely derived from the breakdown of ATP during conditions of energetic crisis, cell swelling, and acidosis, affects cerebral blood flow mostly via activation of adenosine A2A receptors in balancing blood flow with metabolism. The primary effect of stimulating A2A receptors is activation of KATP and KCa channels resulting in smoothmuscle relaxation and elevated blood flow rates.50 Further, it has been shown that inhibition of ADK increases cerebral blood flow (CBF) by augmenting interstitial adenosine levels.56 In addition, chronically increased levels of adenosine have been shown to stimulate angiogenesis.1,29,49 Consequently, chronic overexpression of ADK, as seen in epilepsy, may result in reduced cerebral blood flow (CBF) and reduction of local vasculature. Therefore, ADK-dependent changes in vasculature might provide a diagnostic opportunity for the detection of an epileptogenic focus that is characterized by overexpression of ADK.

This study was designed to assess whether overexpression of ADK links to neuronal hyperexcitability and seizure activity in the neocortex, as well as to develop a diagnostic method to identify cortical areas of neuronal hyperexcitability. Compared with TLE, the pathophysiology and mechanisms for seizure generation in neocortical epilepsy (NCE) are understudied. In particular, a scarcity of clinically relevant rodent models for NCE has limited research progress.33,45,48 In addition, NCE remains one of the most difficult forms of epilepsy to treat, and there is a need for improved diagnostic methods that account for pathophysiological mechanisms of NCE.20,54,66

Since astroglial ADK regulates neuronal excitability of the hippocampus as well as microcirculation, we hypothesized that a focal disruption of adenosine homeostasis induced by overexpression of ADK in astrocytes in the neocortex might be sufficient to induce neuronal hyperexcitability and changes in microcirculation. We used adeno-associated virus 8 (AAV8)–based viral vectors to induce overexpression of ADK in astrocytes of the neocortex.57 To demonstrate ADK-related changes in microcirculation we used a novel imaging technology, optical microangiography (OMAG),30–32 which is based on endogenous light scattering from biological tissue. This noninvasive technology allows generation of microstructural and functional vascular images that can resolve the 3D distribution of dynamic blood perfusion at the capillary level resolution in vivo.30–32 Here, we evaluated OMAG as a potential diagnostic tool for the detection of microcirculation changes in the vicinity of focal areas with disrupted adenosine homeostasis. Our data demonstrate that focal overexpression of ADK in astrocytes of the neocortex is sufficient to elicit electrographic seizures with concomitant changes in microcirculation. Our findings are of significance for the understanding of mechanisms of seizure generation in NCE and demonstrate the diagnostic value of the noninvasive OMAG approach to identify focal neocortical areas of hyperexcitability.

Methods

Animals

All animal procedures were performed in a facility accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care and were performed in accordance with protocols approved by the institutional animal care and use committees of the Legacy Research Institute and the Oregon Health & Science University and according to principles outlined in the National Institutes for Health Guide for the Care and Use of Laboratory Animals. A total of 16 C57BL/6 male mice (20–30 g) were subjected to neocortical virus injection followed by OMAG and EEG (electroencephalography) (Fig. 1A). All animals were group-housed with water and food available ad libitum in temperature- and humidity-controlled rooms with a 12-hour light/dark cycle throughout the experimental period.

Fig. 1.
Fig. 1.

Overexpression of ADK in the neocortex of mice induced by unilateral injection of ADK-SS virus. A: Schematic illustration of the experimental paradigm. Mice were first subjected to unilateral intracortical virus injection (Virus inj.), followed after 8 weeks by an OMAG scan and cortical EEG recordings (EEG rec.). After completion of the EEG, animals were euthanized for immunohistochemical analysis (IHC). B: Schematic illustration of the virus-transduced area (Virus inj.) and locations of the EEG recording electrodes (red) in frontal cortex (Frontal rec.), focal area of ADK overexpression (Focal rec.), and above the cerebellum (reference recording [Ref. rec.]). The area scanned by OMAG is indicated by a dotted green line. C: Representative ADK immunoperoxidase (upper panel) and Nissl (lower panel) staining of brain slices showing the contralateral (contra) or ipsilateral (ipsi) injected (ADK-SS) hemisphere of an ADK-SS recipient and the virus-injected hemisphere of an AAV-Null–injected control animal (AAV-Null). Bar = 500 μm. The bar graph demonstrates relative densities of ADK immunoreactivity of DAB-stained brain sections of ADK-SS or AAV-Null virus–injected mice (2 sections per animal, 5 animals per group). D: Representative ADK staining of consecutive brain slices from an ADK-SS virus–injected animal showing the 3D extent of ADK overexpression. The notch (arrow) on the −2.15-mm slice indicates the locus of the electrode implantation. Bar = 1 mm. Data are displayed as mean ± SEM. #p < 0.01, ADK-SS versus AAV-Null group; *p < 0.01, ipsilateral versus contralateral side.

Adeno-Associated Virus Production and Delivery

As described previously,57,60 the short cytoplasmic isoform of ADK was introduced into an adeno-associated virus (AAV)–based system in which ADK cDNA was placed under the control of the astrocyte-specific gfaABC1D promoter.37 This vector is designated as ADKSS. An AAV8-pGfa-null (AAV-Null) virus containing an empty vector backbone was used as a negative control vector. Virus production and titer determination was performed as described previously.57,60 For virus delivery, the ADK-SS or AAV-Null virus was unilaterally injected into the right side of the neocortex under general anesthesia (1.5% isoflurane, 70% N2O, and 28.5% O2), in a volume of 0.5 μl of concentrated viral solution (1012 genomic particles/ml), with the following stereotactic coordinates: anteroposterior (AP) = −2.10 mm; mediolateral (ML) = –1.50 mm; dorsoventral (DV) = –0.80 mm. After the cortical injections, the incisions were sutured and treated with an antiseptic agent, and the animals were then returned to their home cages.

Optical Microangiography

To test whether focal disruption of adenosine homeostasis affects cortical microcirculation, CBF was measured in mice 8 weeks after viral delivery, using OMAG, a high-resolution optical coherence tomography technique that is capable of producing 3D images of dynamic blood perfusion within microcirculatory tissue beds at an imaging depth up to 2 mm below the surface.31 For example, OMAG is able to measure changes of cerebral blood perfusion in response to systemic hypoxia and hyperoxia.31 Since OMAG requires intact cerebral cortex, this step was performed before the EEG recordings (Fig. 1A). Prior to imaging, the mouse was immobilized in a custom-made stereotactic stage under general anesthesia (1.5% isoflurane, 70% N2O, and 28.5% O2) and the mouse's head was shaved and depilated. The body temperature was maintained between 35.5°C and 36.5°C with a thermostat-controlled heating pad (Harvard Apparatus). An incision of 1 cm was made along the sagittal suture, and the frontal parietal and interparietal bones were exposed by pulling the skin to the sides. The animal was then positioned under the OMAG scanning probe. To acquire microvascular images over a large area of the cortex, scanning was performed clockwise, which resulted in 6–8 OMAG images per mouse, covering both hemispheres over an area of approximately 4 × 5 mm (Fig. 1B). The total imaging time per mouse was less than 10 minutes. After imaging and data acquisition, blood flow signals were isolated from each 3D OMAG data set using a volume segmentation algorithm.61–63 The volume segmentation algorithm was applied to each OMAG data set to isolate blood flow signals within the cortex. A maximum projection method was then used to project the blood flow signals into the x-y plane to reduce the image size. The final OMAG image, including the area of the viral injection site and the corresponding area in the contralateral hemisphere, was obtained by assembling 6 individual images (or 8 images, if necessary). The cerebral blood perfusion was estimated by computing the functional vessel density in the area of interest in the final OMAG image.53 To achieve this, the OMAG image was first converted to a binary flow map by setting a fixed intensity threshold. A circular area with a diameter of 2 mm centered at the viral injection site was identified on the flow map. The percentage of pixels with a binary value of 1 versus pixel numbers of the whole area was calculated as the estimation of vessel density (the quantitative equivalence of cerebral blood perfusion).53 Cerebral blood perfusion in the corresponding area in the contralateral hemisphere was estimated using the same method. Statistical comparisons of vessel density between the ADK-SS virus–injected animals and the control animals (injected with AAV-Null virus) were then undertaken. Additional comparisons were made with the virus-injected sites and their corresponding contralateral locations. The processing and analysis on the OMAG images was carried out using MATLAB programming software (MathWorks, Inc.).

Electroencephalography

Three days following the OMAG scan, bipolar, coated stainless steel electrodes (80 μm in diameter, Plastics One) were implanted into the virus-injected cortex (coordinates: AP = −2.1 mm; ML = −1.5 mm; DV = −1.0 mm, with bregma as reference) under general anesthesia. A cortical screw electrode (AP = 1.0 mm; ML = 1.5 mm) and a ground reference electrode (AP = 6.0 mm; ML = 0 mm) were placed over the frontal cortex and cerebellum, respectively (Fig. 1B). All electrodes were secured to the skull with dental cement. One week later, EEG monitoring was performed as previously described60 with modifications as follows. After the mice were habituated to the EEG-recording procedure for 4 hours, EEG recording was performed for an additional 24 hours. Quantification of the EEG data was performed by an investigator who was blinded to the experimental treatment. An electrographic seizure was defined as high-amplitude rhythmic discharge that clearly represented a new pattern of tracing lasting for more than 5 seconds (repetitive spikes, spike-and-wave discharges, or slow waves). Epileptic events occurring with an interval of less than 5 seconds without the EEG returning to baseline were defined as belonging to the same seizure. Seizures were primarily electrographic in nature, although frequently accompanied by arrest or staring episodes; no concurrent convulsions were observed. Seizure quantification was performed exclusively on the basis of intracortical EEG recordings.

Immunohistochemistry

To determine changes in ADK expression, virus-injected mice were euthanized after EEG evaluation for immunohistochemical analysis as previously described.57 For the detection of ADK, a primary anti–ADK antibody (1:5000)59 was used. To detect the cell-type specificity of ADK expression,57 double immunofluorescence staining was performed using a combination of primary antibodies directed against ADK (polyclonal, rabbit) and glial fibrillary acidic protein (GFAP; monoclonal, mouse 1:400, MAB360, Chemicon International), or neuronal nuclei (NeuN; monoclonal, mouse 1:750, MAB377, Chemicon). Nissl staining was performed separately on adjacent brain slices. Digital images were acquired using a Zeiss AxioPlan inverted microscope equipped with an AxioCam 1Cc1 camera (Carl Zeiss MicroImaging). To quantitatively evaluate the expression level of ADK, the optical density of ADK-positive staining was measured using Image-Pro Plus software (version 5.1, Media Cybernetics, Inc.) as previously described,40 using 1 coronal brain section at the level of the virus-injection from each animal. Data were normalized to the contralateral cortical ADK expression levels.

Statistical Analysis

Data are expressed as means ± standard error of the mean (SEM). Statistical analysis was performed by means of a 1-way ANOVA followed by Bonferroni post hoc analysis or a Student t-test. A p value < 0.05 was considered statistically significant.

Results

Focal Overexpression of ADK in Astrocytes Within the Neocortex

To determine changes in ADK expression, 8 weeks following virus injection and after completion of OMAG imaging and EEG recordings (Fig. 1A), animals were euthanized and their brains evaluated histologically. Nissl staining to assess general brain morphology did not reveal any gross morphological deviations in virus-injected animals compared with controls; in particular, virus injection was not associated with any overt signs of pyknosis, karyolysis, or shrunken cell bodies (Fig. 1C, lower panel). The lack of any obvious virus-induced cytotoxic effects is in line with the demonstrated safety profile of the AAV system for CNS applications.64,65 Immunohistochemical data demonstrated that the cortical injection of the ADKSS virus induced a focal overexpression of ADK within the injected neocortex (Fig. 1C), whereas injection of the AAV-Null virus had no obvious effects on ADK expression patterns, which were comparable to those seen in the noninjected contralateral cortex (Fig. 1C). Quantitative analysis of immunodensities demonstrated that the ADKSS virus induced a robust focal increase in ADK expression, which showed a mean fold increase of 4.8 ± 0.86 compared with ADK expression levels in the contralateral hemisphere (p < 0.01, 5 mice per group) and a mean fold increase of 4.4 ± 0.77 compared with the AAV-Null–injected hemisphere (p < 0.01, 5 mice per group) (Fig. 1C). Conversely, AAV-Null virus injection did not cause significant changes in ADK expression compared with the contralateral hemisphere (p > 0.05, 5 mice per group) (Fig. 1C). We previously demonstrated in a transgenic model system that a 63.6% increase in ADK resulted in a 50% reduction in the ambient tone of adenosine;57 we therefore conclude that the focal overexpression of ADK achieved here triggers a local deficiency in adenosine. To identify the rostral-caudal extent of ADK-SS–induced ADK overexpression, we investigated a series of brain slices spanning the region from −1.70 to −2.75 mm from bregma (Fig. 1D). Analysis of the extent of virus-induced ADK overexpression demonstrated a focal area of high levels of ADK within the neocortex with a diameter about 1.9 ± 0.4 mm (5 mice).

Higher-resolution images of ADK immunoperoxidase–stained sections demonstrated profound differences in the subcellular localization of ADK between experimental groups (Fig. 2A). Images from ADK-SS–injected cortex showed high levels of ADK expression in cell somata and cellular processes, consistent with overexpression of the cytoplasmic isoform of ADK as expressed by the virus. ADK-positive cells had characteristic morphological features of astrocytes, with a star-like appearance and several primary processes originating from the soma (Fig. 2A, left, inset). In contrast, ADK expression in control samples from AAV-Null animals had a characteristic pattern of prominent nuclear ADK expression coupled with a diffuse homogenous staining of ADK throughout the tissue at much lower levels, in line with our previous characterization of astrocytic ADK expression in naïve mice.22,59 To confirm the cell-type specificity of ADK-SS–induced ADK overexpression, we performed a double immunofluorescence analysis by costaining ADK with either GFAP or NeuN (Fig. 2B and 2C). As expected, and in line with findings from our previous work,57,60 ADK expression was colocalized with GFAP expression and was confined to GFAP-positive cells (Fig. 2B, center, left panel) and not seen in NeuN-positive cells (Fig. 2B, center, right panel). Higher-resolution images confirmed the differential expression pattern of ADK-SS–induced overexpression of exogenous ADK (cytoplasm) versus endogenous ADK (nucleus) (Fig. 2C). Together, our data demonstrate a robust focal overexpression of the cytoplasmic isoform of ADK in cortical astrocytes from ADKSS–injected mice. Importantly, overexpression of ADK was confined to a distinct focal area, with a diameter of about 2 mm, close to the cortical surface, whereas other brain areas were not affected by ADK-SS or a control virus injection.

Fig. 2.
Fig. 2.

Cellular expression pattern of ADK. A: Results of ADK immunohistochemistry on coronal sections from mouse brain injected with ADK-SS virus or AAV-Null virus. Bar = 25 μm. B: Immunofluorescence of ADK-SS virus–induced ADK expression (red) co-stained with antibodies directed against GFAP (green, in center left panel) or NeuN (green, in center right panel). Bar = 25 μm. C: Double-immunofluorescence staining of ADK (red) and GFAP (green) at higher resolution showing expression of endogenous ADK within the nucleus and cell soma of astrocytes from the neocortex of an AAV-Null–injected control animal (upper) and expression of exogenous ADK throughout the soma and processes of astrocytes from an ADK-SS recipient (lower). Bar = 10 μm.

Recurrent Electrographic Seizures

To evaluate whether focal neocortical overexpression of ADK influenced neuronal excitability, we performed continuous EEG monitoring of neocortical electrographic activity, 8 to 9 weeks after intracortical injection of ADKSS or AAV-Null virus. EEG recordings from the neocortical injection site illustrated that ADK-SS virus–injected mice developed spontaneous recurrent electrographic seizures within the neocortex (Fig. 3A, lower panel) whereas AAV-Null virus–injected animals were devoid of any seizure activity (Fig. 3A, upper panel). The neocortical electrographic seizures were characterized by a gradual increase in amplitude that became rhythmic at the beginning of the seizure (Fig. 3A, lower panel, closed arrow) whereas the end of the seizure had an overt drop in amplitude (Fig. 3C, lower panel, open arrow). The neocortical seizures, which occurred in the ADK-SS–injected animals had a seizure frequency of 1.3 ± 0.2 seizures per hour (n = 8) with an average duration of 94 ± 8 seconds. It is important to note that, even though spontaneous electrographic seizures were observed in all animals with ADK overexpression, those activities were not associated with any convulsive behavior. Power spectral analysis of the single event depicted in Fig. 3A indicates a predominant ictal frequency of around 3 Hz (Fig. 3C), which is in contrast to interictal EEG activity from ADK-SS recipients or baseline activity in AAV-Null recipients (Fig. 3B). Together, the EEG data demonstrated that focal viral overexpression of ADK within astrocytes was sufficient to trigger spontaneous recurrent neocortical seizures in the absence of any epileptogenic event.

Fig. 3.
Fig. 3.

Neocortical electroencephalographic activity of AAV-injected mice. A: Focal intracortical EEG traces from the neocortical region of mice injected with AAV-Null (upper panel) or ADK-SS (lower panel). The lower traces are at higher resolution and represent background activity (AAV-Null) and a complete representative seizure (ADK-SS), including seizure onset (closed arrow) and end (open arrow). B and C: Power spectral analysis of EEG activity from mice injected with AAV-Null (B) or ADK-SS (C). h = hours; s = seconds.

Reduced Focal Vessel Density

To evaluate whether disruption of adenosine homeostasis causes changes in microcirculation, we employed the OMAG approach to image dynamic in vivo cerebral blood perfusion and vasculature changes in the mice 8 weeks after viral injection. The high-contrast OMAG images demonstrated well-resolved cerebral vasculature indicating the cerebral perfusion status of the mice (Fig. 4A). OMAG data showed that the AAV-Null virus injection did not cause focal changes in cerebral blood perfusion and vessel density similar to the contralateral hemisphere (Fig. 4A, left panel). Importantly, OMAG images showed that ADK-SS–induced overexpression of ADK resulted in an overt reduced local cerebral vessel density indicating reduced cerebral blood perfusion surrounding the viral injection site (Fig. 4A, right panel). The resulting quantitative data demonstrated a significant reduction of focal cerebral blood perfusion in the ipsilateral neocortex of ADK-SS recipients compared with the corresponding region in AAV-Null recipients (23% reduction of CBF, p < 0.05, n = 5, Fig. 4B, left panel). Likewise, the focal vessel density in the ipsilateral neocortex of ADK-SS–injected animals was also significantly lower (29% reduction) compared with the contralateral hemisphere of the same animal (29% reduction of vessel density, p < 0.05, n = 5, Fig. 4B, right panel). Together, the analysis of the OMAG data indicated that disruption of adenosine homeostasis by overexpression of ADK led to a reduction in focal cerebral blood perfusion and cerebral vessel density.

Fig. 4.
Fig. 4.

OMAG of cerebral blood perfusion and vessel densities in AAV-injected mice. A: Representative 2D images of cortical vessel density at capillary-level resolution in AAV-Null or ADK-SS virus recipients. The asterisks indicate the coordinate of virus injection and the dashed lines indicate the virus-affected area. Bar = 1 mm. B: Quantitative analysis showing reduced cortical vessel density in the hemispheres injected with ADK-SS, compared with those injected with AAV-Null (left panel) or compared with those in the contralateral hemisphere (right panel). Cortical vessel densities are shown as percentage relative to control. Data are displayed as mean ± SEM. #p < 0.05 paired comparisons t-test (n = 5 per group).

Discussion

To better understand mechanisms of ictogenesis in NCE, we report here a novel animal model of NCE with neocortical overexpression of ADK in astrocytes, and demonstrate the feasibility of OMAG as an advanced noninvasive imaging tool to detect the epileptic focus in this model. We demonstrate the following key findings: 1) virus-induced overexpression of astrocytic ADK in neocortex is sufficient to produce robust spontaneous recurrent focal electrographic seizures at a frequency of 1.3 ± 0.2 seizures per hour, and 2) the seizure-generating focus spatially aligns with overexpression of ADK and detectable microcirculation changes, which can be evaluated noninvasively by OMAG.1,29,47,49–51,56

ADK is the key enzyme for the metabolic clearance of adenosine and exists in 2 isoforms, which are specific for the nucleus (ADK long [ADK-L]) or the cytoplasm (ADK short [ADK-S]).8 The specific roles of the different isoforms have not yet been fully elucidated, but mounting evidence suggests that nuclear ADK-L plays a role in epigenetic functions and cell proliferation, whereas the cytoplasmic ADK-S appears to be responsible for the metabolic clearance of extracellular adenosine.8

As our current findings validate (Fig. 2A), and as previously demonstrated,22,59 under control or baseline conditions, the majority of ADK expression is confined to the nucleus or perinuclear area of astrocytes. However, in the epileptic brain, astrocytic ADK is not only increased in quantity, but also in subcellular distribution, with a robust shift of ADK expression from the perinuclear area into the network of astrocyte processes.40 Thus quantity and distribution of the cytoplasmic isoform of ADK might play a key role in ictogenesis. We therefore induced overexpression of the cytoplasmic isoform of ADK in astrocytes, obtaining profound expression of ADK in astrocytic processes (Fig. 2C) similar to ADK expression patterns in a mouse model of kainic acid induced–epilepsy.40 Our demonstration that overexpression of the cytoplasmic isoform of ADK in astrocytes is sufficient to trigger focal neocortical seizures further supports a role of ADK located in astrocyte processes for ictogenesis.

While AAV-induced overexpression of ADK within astrocytes was sufficient to trigger spontaneous electrographic seizures in the neocortex, none of the mice in this study exhibited any signs of convulsive seizures. Periods of immobility or staring episodes were occasionally associated with the electrographic seizures, indicating a phenotype of partial epilepsy without generalization. The hourly seizure rate, as well as the brief duration of the seizures (94 seconds) and the low intra-ictal spike frequency (3 Hz), is in line with electrographic seizure patterns that have been linked to deficient adenosine signaling. Focal CA3 selective seizures (4.3 seizures per hour; 17.5 seconds' duration), linked to astrogliosis and overexpression of ADK after intra-amygdaloid kainic acid injection, spontaneous seizures in Adk-tg mice with brain-wide overexpression of ADK (4.8 seizures per hour; 26.7 seconds' duration), and spontaneous seizures in adenosine A1R knockout mice (5.3 seizures per hour; 27.8 seconds' duration) all share the same characteristic seizure pattern with an intra-ictal spike frequency of 3–4 Hz.38 Together these findings suggest that overexpression of ADK, resulting in deficient signaling through the adenosine A1 receptor, triggers a characteristic seizure pattern also found in focal seizure models that result from an epilepsy-precipitating status epilepticus. Since ADK expression was found to be focally restricted to a sphere with a diameter of about 2 mm (Fig. 1), adenosine homeostasis in the remaining brain is not likely to be compromised by this manipulation. The lack of seizure spread in this model is consistent with previous findings demonstrating that adenosine A1 receptor activation by endogenous adenosine is a prerequisite for the prevention of seizure spread.18,39 Thus, a local deficiency of adenosine caused by the cell-type–selective overexpression of ADK in astrocytes, triggers focal electrographic seizures by insufficient activation of the A1 receptor, which normally limits neuronal excitability by mediating presynaptic inhibition and by stabilizing the postsynaptic membrane potential. The focal nature of our manipulation is further supported by OMAG imaging suggesting a tight spatial match between overexpression of ADK and changes in the microvasculature.

Adenosine is one of the most potent vasodilators in different kinds of tissues and organs. Under both physiological and pathophysiological conditions, extracellular adenosine provokes acute local vasodilation within the cerebral microcirculature,47 mainly via activation of the cAMP (cyclic adenosine monophosphate) pathway through adenosine A2A and A2B receptors.49–51 Studies also suggest that adenosine exerts long-term effects on microvasculature via facilitation of angiogenesis1 through the induction of angiogenic growth factors such as vascular endothelial growth factor.1 Whereas the application of adenosine or pharmacological agents that alter adenosine metabolism can stimulate vasodilation and blood vessel growth, the inhibition of ADK can increase CBF through an increase in interstitial adenosine levels.56

While not directly investigated here due to space considerations, we have demonstrated previously that the overexpression of astrocytic ADK leads to a robust reduction in ambient adenosine levels,15,57 whereas down-regulation of endogenous ADK is a physiological response to raise adenosine levels under conditions of metabolic or energetic stress.52 The reduced vessel density and reduced cerebral blood perfusion demonstrated here (Fig. 4) are consistent with a reduced concentration of adenosine in the neocortical focus of overexpressed ADK and reduced activation of the vascular A2A receptor, which plays an important role in the regulation of CBF and angiogenesis.50 Given the lower sensitivity of the A2A receptor compared with the A1 receptor, even a minor reduction in adenosine is likely to affect the vasculature. In addition, seizures by themselves are known to trigger the release of adenosine as an endogenous mechanism contributing to seizure termination,16,35 and seizure-induced adenosine release is consistent with an increase of CBF during ictal seizure events.21,34 Our findings of consistently reduced CBF within the ADK-related seizure focus suggest that seizure-induced elevations of adenosine are subject to enhanced metabolic clearance through increased levels of ADK and are therefore no longer effective in influencing CBF. Astrocytes play a central role in the vasomotor response, in which neurovascular coupling links massive Ca2+ elevations in the ictal phase to changes in CBF.21 However, a reduced basal adenosine level may lead to a loss of postictal cerebral vasodilation.14,47 Given the frequent occurrence and long duration of ADK-induced neocortical electrographic seizures, it is likely that the net effects of the CBF measurement obtained by OMAG are based on a combination of ictal and interictal events.

Clinically, neocortical epileptogenic brain areas may be resected following presurgical evaluation through a variety of techniques:20 1) computerized EEG source imaging, based on surface or invasive intracranial EEG, targets focal abnormal epileptic activity;44 2) neuroimaging approaches, such as PET, SPECT, or functional MRI are used to detect regional cerebral blood volumes;27,28,46 and 3) CT and MRI are used to detect epilepsy-related dysplasias.10 However, each of the above approaches has its own set of limitations in diagnostic sensitivity and predictive value.36 Alternative or additional diagnostic approaches might be useful to improve planning of a resection4,58 and are in particular need for NCE, since this condition in general shows a lower rate of concordance between diagnostic modalities as compared with mesial TLE.36 Changes in neuronal activities are known to alter the light scattering and absorption properties of brain tissue. The intrinsic optical signal imaging technique provides 2D cortical maps of these changes and has been used to study the functional organization of visual cortex23,42 and to perform intraoperative mapping of functional and epileptiform activities in human subjects.11,24–26,55 These 2D techniques are only able to detect hemodynamic changes on the cortical surface with limited spatial resolution. The novel imaging technology OMAG investigated here is based on endogenous light scattering from biological tissue to obtain microstructural and functional vascular images, and it can resolve the 3D distribution of dynamic blood perfusion at capillary-level resolution within the microcirculatory bed in vivo.30 In the present study, we demonstrate that OMAG is able to detect a region with changes of microvasculature and blood perfusion in a diameter of 2 mm, which reflects the area with overexpression of virus-induced ADK. This finding indicates that a potentially small neocortical epileptic focus might be detectable with OMAG, a technology that is noninvasive and reproducibly delivers high-resolution (100-μm) images without the need of exogenous contrasting agents, as usually needed for CT, MRI, or PET.61 We show here that OMAG can reliably detect focal overexpression of ADK. Since overexpression of ADK appears to be a common pathological hallmark found in many different forms of experimental and clinical epilepsy,2,3,13,22,39,40,43 OMAG could represent a novel diagnostic tool to identify focal areas of increased ADK expression as possible predictors for epileptogenic zones.

Conclusions

Findings from this mouse model suggest that focal adenosine deficiency is sufficient to generate a neocortical focus of hyperexcitability, which is also characterized by reduced vessel density. The authors conclude that their model constitutes a useful tool to study NCEs and that OMAG constitutes a noninvasive diagnostic tool for imaging seizure foci with disrupted adenosine homeostasis.

Acknowledgments

We thank Hrebesh M. Subhash and Brian Anderson for their technical support and Shirley McCartney, Ph.D., for editorial assistance.

Disclosure

This work was supported by Grant R01 NS061844 (to Dr. Boison) from the National Institutes of Health. Dr. Sun is in receipt of funds from the Congress of Neurological Surgeons' Christopher C. Getch Flagship Fellowship Award. None of the authors has any conflict of interest to disclose.

Author contributions to the study and manuscript preparation include the following. Conception and design: Boison, Shen, Sun. Acquisition of data: Boison, Shen, Sun, Zhi, Lan, Wang. Analysis and interpretation of data: all authors. Drafting the article: all authors. Critically revising the article: all authors. Reviewed submitted version of manuscript: all authors. Statistical analysis: Shen, Sun.

A portion of this work was presented at the 80th Annual Scientific meeting of the American Association of Neurological Surgeons, April 14–18, 2012, in Miami, Florida, and was featured in the meeting press release.

References

  • 1

    Adair TH: Growth regulation of the vascular system: an emerging role for adenosine. Am J Physiol Regul Integr Comp Physiol 289:R283R2962005

  • 2

    Aronica ESandau USIyer ABoison D: Glial adenosine kinase—a neuropathological marker of the epileptic brain. Neurochem Int 63:6886952013

  • 3

    Aronica EZurolo EIyer Ade Groot MAnink JCarbonell C: Upregulation of adenosine kinase in astrocytes in experimental and human temporal lobe epilepsy. Epilepsia 52:164516552011

  • 4

    Blume WTGanapathy GRMunoz DLee DH: Indices of resective surgery effectiveness for intractable nonlesional focal epilepsy. Epilepsia 45:46532004

  • 5

    Boison D: Adenosine augmentation therapies (AATs) for epilepsy: prospect of cell and gene therapies. Epilepsy Res 85:1311412009

  • 6

    Boison DAdenosine augmentation therapy. Noebels JLAvoli MRogawski MA: Jasper's Basic Mechanisms of the Epilepsies ed 4OxfordOxford University Press2012. 11501160

  • 7

    Boison D: Adenosine dysfunction in epilepsy. Glia 60:123412432012

  • 8

    Boison D: Adenosine kinase: exploitation for therapeutic gain. Pharmacol Rev 65:9069432013

  • 9

    Boison DChen JFFredholm BB: Adenosine signaling and function in glial cells. Cell Death Differ 17:107110822010

  • 10

    Bulakbaşi NBozlar UKarademir IKocaoğlu MSomuncu I: CT and MRI in the evaluation of craniospinal involvement with polyostotic fibrous dysplasia in McCune-Albright syndrome. Diagn Interv Radiol 14:1771812008

  • 11

    Cannestra AFPouratian NBookheimer SYMartin NABeckerand DPToga AW: Temporal spatial differences observed by functional MRI and human intraoperative optical imaging. Cereb Cortex 11:7737822001

  • 12

    Coney AMMarshall JM: Contribution of adenosine to the depression of sympathetically evoked vasoconstriction induced by systemic hypoxia in the rat. J Physiol 549:6136232003

  • 13

    de Groot MIyer AZurolo EAnink JHeimans JJBoison D: Overexpression of ADK in human astrocytic tumors and peritumoral tissue is related to tumor-associated epilepsy. Epilepsia 53:58662012

  • 14

    DiGeronimo RJGegg CAZuckerman SL: Adenosine depletion alters postictal hypoxic cerebral vasodilation in the newborn pig. Am J Physiol 274:H1495H15011998

  • 15

    Diógenes MJNeves-Tomé RFucile SMartinello KScianni MTheofilas P: Homeostatic control of synaptic activity by endogenous adenosine is mediated by adenosine kinase. Cereb Cortex [epub ahead of print]2012

  • 16

    During MJSpencer DD: Adenosine: a potential mediator of seizure arrest and postictal refractoriness. Ann Neurol 32:6186241992

  • 17

    Etherington LAPatterson GEMeechan LBoison DIrving AJDale N: Astrocytic adenosine kinase regulates basal synaptic adenosine levels and seizure activity but not activity-dependent adenosine release in the hippocampus. Neuropharmacology 56:4294372009

  • 18

    Fedele DELi TLan JQFredholm BBBoison D: Adenosine A1 receptors are crucial in keeping an epileptic focus localized. Exp Neurol 200:1841902006

  • 19

    Fredholm BB: Rethinking the purinergic neuron-glia connection. Proc Natl Acad Sci U S A 109:591359142012

  • 20

    Gelziniene GEndziniene MVaiciene NMagistris MRSeeck M: Presurgical evaluation of epilepsy patients. Medicina (Kaunas) 44:5855922008

  • 21

    Gómez-Gonzalo MLosi GBrondi MUva LSato SSde Curtis M: Ictal but not interictal epileptic discharges activate astrocyte endfeet and elicit cerebral arteriole responses. Front Cell Neurosci 5:82011

  • 22

    Gouder NScheurer LFritschy JMBoison D: Overexpression of adenosine kinase in epileptic hippocampus contributes to epileptogenesis. J Neurosci 24:6927012004

  • 23

    Grinvald A: Optical imaging of architecture and function in the living brain sheds new light on cortical mechanisms underlying visual perception. Brain Topogr 5:71751992

  • 24

    Haglund MMHochman DW: Furosemide and mannitol suppression of epileptic activity in the human brain. J Neurophysiol 94:9079182005

  • 25

    Haglund MMHochman DW: Optical imaging of epileptiform activity in human neocortex. Epilepsia 45:Suppl 443472004

  • 26

    Haglund MMOjemann GAHochman DW: Optical imaging of epileptiform and functional activity in human cerebral cortex. Nature 358:6686711992

  • 27

    Haughton VBiswal B: Clinical application of basal regional cerebral blood flow fluctuation measurements by FMRI. Adv Exp Med Biol 454:5835901998

  • 28

    Henry TR: PET: cerebral blood flow and glucose metabolism—presurgical localization. Adv Neurol 83:1051202000

  • 29

    Hong KWShin HKKim HHChoi JMRhim BYLee WS: Metabolism of cAMP to adenosine: role in vasodilation of rat pial artery in response to hypotension. Am J Physiol 276:H376H3821999

  • 30

    Jia YLi PDziennis SWang RK: Responses of peripheral blood flow to acute hypoxia and hyperoxia as measured by optical microangiography. PLoS ONE 6:e268022011

  • 31

    Jia YLi PWang RK: Optical microangiography provides an ability to monitor responses of cerebral microcirculation to hypoxia and hyperoxia in mice. J Biomed Opt 16:0960192011

  • 32

    Jia YQin JZhi ZWang RK: Ultrahigh sensitive optical microangiography reveals depth-resolved microcirculation and its longitudinal response to prolonged ischemic event within skeletal muscles in mice. J Biomed Opt 16:0860042011

  • 33

    Kondo SNajm IKunieda TPerryman SYacubova KLüders HO: Electroencephalographic characterization of an adult rat model of radiation-induced cortical dysplasia. Epilepsia 42:122112272001

  • 34

    Kuhl DEEngel J JrPhelps MESelin C: Epileptic patterns of local cerebral metabolism and perfusion in humans determined by emission computed tomography of 18FDG and 13NH3. Ann Neurol 8:3483601980

  • 35

    Lado FAMoshé SL: How do seizures stop?. Epilepsia 49:165116642008

  • 36

    Lee SKLee SYKim KKHong KSLee DSChung CK: Surgical outcome and prognostic factors of cryptogenic neocortical epilepsy. Ann Neurol 58:5255322005

  • 37

    Lee YMessing ASu MBrenner M: GFAP promoter elements required for region-specific and astrocyte-specific expression. Glia 56:4814932008

  • 38

    Li TLan JQFredholm BBSimon RPBoison D: Adenosine dysfunction in astrogliosis: cause for seizure generation?. Neuron Glia Biol 3:3533662007

  • 39

    Li TLytle NLan JQSandau USBoison D: Local disruption of glial adenosine homeostasis in mice associates with focal electrographic seizures: a first step in epileptogenesis?. Glia 60:83952012

  • 40

    Li TRen GLusardi TWilz ALan JQIwasato T: Adenosine kinase is a target for the prediction and prevention of epileptogenesis in mice. J Clin Invest 118:5715822008

  • 41

    Li TSteinbeck JALusardi TKoch PLan JQWilz A: Suppression of kindling epileptogenesis by adenosine releasing stem cell-derived brain implants. Brain 130:127612882007

  • 42

    Martin KA: Microcircuits in visual cortex. Curr Opin Neurobiol 12:4184252002

  • 43

    Masino SALi TTheofilas PSandau USRuskin DNFredholm BB: A ketogenic diet suppresses seizures in mice through adenosine A1 receptors. J Clin Invest 121:267926832011

  • 44

    Michel CMMurray MMLantz GGonzalez SSpinelli LGrave de Peralta R: EEG source imaging. Clin Neurophysiol 115:219522222004

  • 45

    Najm IMTilelli CQOghlakian R: Pathophysiological mechanisms of focal cortical dysplasia: a critical review of human tissue studies and animal models. Epilepsia 48:Suppl 221322007

  • 46

    Neirinckx RD: Evaluation of regional cerebral blood flow with 99mTc-d, 1 HM-PAO and SPECT. Neurosurg Rev 10:1811841987

  • 47

    O'Regan MEBrown JK: Abnormalities in cardiac and respiratory function observed during seizures in childhood. Dev Med Child Neurol 47:492005

  • 48

    Oghlakian ROTilelli CQHiremath GKAlexopoulos AVNajm IM: Single injection of a low dose of pentylenetetrazole leads to epileptogenesis in an animal model of cortical dysplasia. Epilepsia 50:8018102009

  • 49

    Pelligrino DAVetri FXu HL: Purinergic mechanisms in gliovascular coupling. Semin Cell Dev Biol 22:2292362011

  • 50

    Phillis JW: Adenosine and adenine nucleotides as regulators of cerebral blood flow: roles of acidosis, cell swelling, and KATP channels. Crit Rev Neurobiol 16:2372702004

  • 51

    Phillis JWLungu CLBarbu DEO'Regan MH: Adenosine's role in hypercapnia-evoked cerebral vasodilation in the rat. Neurosci Lett 365:692004

  • 52

    Pignataro GMaysami SStuder FEWilz ASimon RPBoison D: Downregulation of hippocampal adenosine kinase after focal ischemia as potential endogenous neuroprotective mechanism. J Cereb Blood Flow Metab 28:17232008

  • 53

    Reif RQin JAn LZhi ZDziennis SWang R: Quantifying optical microangiography images obtained from a spectral domain optical coherence tomography system. Int J Biomed Imaging 2012:5097832012

  • 54

    Schuele SULüders HO: Intractable epilepsy: management and therapeutic alternatives. Lancet Neurol 7:5145242008

  • 55

    Schwartz THChen LMFriedman RMSpencer DDRoe AW: Intraoperative optical imaging of human face cortical topography: a case study. Neuroreport 15:152715312004

  • 56

    Sciotti VMVan Wylen DG: Increases in interstitial adenosine and cerebral blood flow with inhibition of adenosine kinase and adenosine deaminase. J Cereb Blood Flow Metab 13:2012071993

  • 57

    Shen HYLusardi TAWilliams-Karnesky RLLan JQPoulsen DJBoison D: Adenosine kinase determines the degree of brain injury after ischemic stroke in mice. J Cereb Blood Flow Metab 31:164816592011

  • 58

    Siegel AMJobst BCThadani VMRhodes CHLewis PJRoberts DW: Medically intractable, localization-related epilepsy with normal MRI: presurgical evaluation and surgical outcome in 43 patients. Epilepsia 42:8838882001

  • 59

    Studer FEFedele DEMarowsky ASchwerdel CWernli KVogt K: Shift of adenosine kinase expression from neurons to astrocytes during postnatal development suggests dual functionality of the enzyme. Neuroscience 142:1251372006

  • 60

    Theofilas PBrar SStewart KAShen HYSandau USPoulsen DJ: Adenosine kinase as a target for therapeutic antisense strategies in epilepsy. Epilepsia 52:5896012011

  • 61

    Wang RK: Three-dimensional optical micro-angiography maps directional blood perfusion deep within microcirculation tissue beds in vivo. Phys Med Biol 52:N531N5372007

  • 62

    Wang RKHurst S: Mapping of cerebro-vascular blood perfusion in mice with skin and skull intact by Optical Micro-AngioGraphy at 1.3 mum wavelength. Opt Express 15:11402114122007

  • 63

    Wang RKJacques SLMa ZHurst SHanson SRGruber A: Three dimensional optical angiography. Opt Express 15:408340972007

  • 64

    Weinberg MSMcCown TJ: Current prospects and challenges for epilepsy gene therapy. Exp Neurol 244:27352013

  • 65

    Weinberg MSSamulski RJMcCown TJ: Adeno-associated virus (AAV) gene therapy for neurological disease. Neuropharmacology 69:82882013

  • 66

    Wykes RCHeeroma JHMantoan LZheng KMacDonald DCDeisseroth K: Optogenetic and potassium channel gene therapy in a rodent model of focal neocortical epilepsy. Sci Transl Med 4:161ra1522012

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Article Information

Address correspondence to: Detlev Boison, Ph.D., RS Dow Neurobiology Laboratories, Legacy Research Institute, 1225 NE 2nd Ave., Portland, OR 97232. email: dboison@downeurobiology.org.

Drs. Shen and Sun contributed equally to this work.

Please include this information when citing this paper: published online November 22, 2013; DOI: 10.3171/2013.10.JNS13918.

© AANS, except where prohibited by US copyright law.

Headings

Figures

  • View in gallery

    Overexpression of ADK in the neocortex of mice induced by unilateral injection of ADK-SS virus. A: Schematic illustration of the experimental paradigm. Mice were first subjected to unilateral intracortical virus injection (Virus inj.), followed after 8 weeks by an OMAG scan and cortical EEG recordings (EEG rec.). After completion of the EEG, animals were euthanized for immunohistochemical analysis (IHC). B: Schematic illustration of the virus-transduced area (Virus inj.) and locations of the EEG recording electrodes (red) in frontal cortex (Frontal rec.), focal area of ADK overexpression (Focal rec.), and above the cerebellum (reference recording [Ref. rec.]). The area scanned by OMAG is indicated by a dotted green line. C: Representative ADK immunoperoxidase (upper panel) and Nissl (lower panel) staining of brain slices showing the contralateral (contra) or ipsilateral (ipsi) injected (ADK-SS) hemisphere of an ADK-SS recipient and the virus-injected hemisphere of an AAV-Null–injected control animal (AAV-Null). Bar = 500 μm. The bar graph demonstrates relative densities of ADK immunoreactivity of DAB-stained brain sections of ADK-SS or AAV-Null virus–injected mice (2 sections per animal, 5 animals per group). D: Representative ADK staining of consecutive brain slices from an ADK-SS virus–injected animal showing the 3D extent of ADK overexpression. The notch (arrow) on the −2.15-mm slice indicates the locus of the electrode implantation. Bar = 1 mm. Data are displayed as mean ± SEM. #p < 0.01, ADK-SS versus AAV-Null group; *p < 0.01, ipsilateral versus contralateral side.

  • View in gallery

    Cellular expression pattern of ADK. A: Results of ADK immunohistochemistry on coronal sections from mouse brain injected with ADK-SS virus or AAV-Null virus. Bar = 25 μm. B: Immunofluorescence of ADK-SS virus–induced ADK expression (red) co-stained with antibodies directed against GFAP (green, in center left panel) or NeuN (green, in center right panel). Bar = 25 μm. C: Double-immunofluorescence staining of ADK (red) and GFAP (green) at higher resolution showing expression of endogenous ADK within the nucleus and cell soma of astrocytes from the neocortex of an AAV-Null–injected control animal (upper) and expression of exogenous ADK throughout the soma and processes of astrocytes from an ADK-SS recipient (lower). Bar = 10 μm.

  • View in gallery

    Neocortical electroencephalographic activity of AAV-injected mice. A: Focal intracortical EEG traces from the neocortical region of mice injected with AAV-Null (upper panel) or ADK-SS (lower panel). The lower traces are at higher resolution and represent background activity (AAV-Null) and a complete representative seizure (ADK-SS), including seizure onset (closed arrow) and end (open arrow). B and C: Power spectral analysis of EEG activity from mice injected with AAV-Null (B) or ADK-SS (C). h = hours; s = seconds.

  • View in gallery

    OMAG of cerebral blood perfusion and vessel densities in AAV-injected mice. A: Representative 2D images of cortical vessel density at capillary-level resolution in AAV-Null or ADK-SS virus recipients. The asterisks indicate the coordinate of virus injection and the dashed lines indicate the virus-affected area. Bar = 1 mm. B: Quantitative analysis showing reduced cortical vessel density in the hemispheres injected with ADK-SS, compared with those injected with AAV-Null (left panel) or compared with those in the contralateral hemisphere (right panel). Cortical vessel densities are shown as percentage relative to control. Data are displayed as mean ± SEM. #p < 0.05 paired comparisons t-test (n = 5 per group).

References

1

Adair TH: Growth regulation of the vascular system: an emerging role for adenosine. Am J Physiol Regul Integr Comp Physiol 289:R283R2962005

2

Aronica ESandau USIyer ABoison D: Glial adenosine kinase—a neuropathological marker of the epileptic brain. Neurochem Int 63:6886952013

3

Aronica EZurolo EIyer Ade Groot MAnink JCarbonell C: Upregulation of adenosine kinase in astrocytes in experimental and human temporal lobe epilepsy. Epilepsia 52:164516552011

4

Blume WTGanapathy GRMunoz DLee DH: Indices of resective surgery effectiveness for intractable nonlesional focal epilepsy. Epilepsia 45:46532004

5

Boison D: Adenosine augmentation therapies (AATs) for epilepsy: prospect of cell and gene therapies. Epilepsy Res 85:1311412009

6

Boison DAdenosine augmentation therapy. Noebels JLAvoli MRogawski MA: Jasper's Basic Mechanisms of the Epilepsies ed 4OxfordOxford University Press2012. 11501160

7

Boison D: Adenosine dysfunction in epilepsy. Glia 60:123412432012

8

Boison D: Adenosine kinase: exploitation for therapeutic gain. Pharmacol Rev 65:9069432013

9

Boison DChen JFFredholm BB: Adenosine signaling and function in glial cells. Cell Death Differ 17:107110822010

10

Bulakbaşi NBozlar UKarademir IKocaoğlu MSomuncu I: CT and MRI in the evaluation of craniospinal involvement with polyostotic fibrous dysplasia in McCune-Albright syndrome. Diagn Interv Radiol 14:1771812008

11

Cannestra AFPouratian NBookheimer SYMartin NABeckerand DPToga AW: Temporal spatial differences observed by functional MRI and human intraoperative optical imaging. Cereb Cortex 11:7737822001

12

Coney AMMarshall JM: Contribution of adenosine to the depression of sympathetically evoked vasoconstriction induced by systemic hypoxia in the rat. J Physiol 549:6136232003

13

de Groot MIyer AZurolo EAnink JHeimans JJBoison D: Overexpression of ADK in human astrocytic tumors and peritumoral tissue is related to tumor-associated epilepsy. Epilepsia 53:58662012

14

DiGeronimo RJGegg CAZuckerman SL: Adenosine depletion alters postictal hypoxic cerebral vasodilation in the newborn pig. Am J Physiol 274:H1495H15011998

15

Diógenes MJNeves-Tomé RFucile SMartinello KScianni MTheofilas P: Homeostatic control of synaptic activity by endogenous adenosine is mediated by adenosine kinase. Cereb Cortex [epub ahead of print]2012

16

During MJSpencer DD: Adenosine: a potential mediator of seizure arrest and postictal refractoriness. Ann Neurol 32:6186241992

17

Etherington LAPatterson GEMeechan LBoison DIrving AJDale N: Astrocytic adenosine kinase regulates basal synaptic adenosine levels and seizure activity but not activity-dependent adenosine release in the hippocampus. Neuropharmacology 56:4294372009

18

Fedele DELi TLan JQFredholm BBBoison D: Adenosine A1 receptors are crucial in keeping an epileptic focus localized. Exp Neurol 200:1841902006

19

Fredholm BB: Rethinking the purinergic neuron-glia connection. Proc Natl Acad Sci U S A 109:591359142012

20

Gelziniene GEndziniene MVaiciene NMagistris MRSeeck M: Presurgical evaluation of epilepsy patients. Medicina (Kaunas) 44:5855922008

21

Gómez-Gonzalo MLosi GBrondi MUva LSato SSde Curtis M: Ictal but not interictal epileptic discharges activate astrocyte endfeet and elicit cerebral arteriole responses. Front Cell Neurosci 5:82011

22

Gouder NScheurer LFritschy JMBoison D: Overexpression of adenosine kinase in epileptic hippocampus contributes to epileptogenesis. J Neurosci 24:6927012004

23

Grinvald A: Optical imaging of architecture and function in the living brain sheds new light on cortical mechanisms underlying visual perception. Brain Topogr 5:71751992

24

Haglund MMHochman DW: Furosemide and mannitol suppression of epileptic activity in the human brain. J Neurophysiol 94:9079182005

25

Haglund MMHochman DW: Optical imaging of epileptiform activity in human neocortex. Epilepsia 45:Suppl 443472004

26

Haglund MMOjemann GAHochman DW: Optical imaging of epileptiform and functional activity in human cerebral cortex. Nature 358:6686711992

27

Haughton VBiswal B: Clinical application of basal regional cerebral blood flow fluctuation measurements by FMRI. Adv Exp Med Biol 454:5835901998

28

Henry TR: PET: cerebral blood flow and glucose metabolism—presurgical localization. Adv Neurol 83:1051202000

29

Hong KWShin HKKim HHChoi JMRhim BYLee WS: Metabolism of cAMP to adenosine: role in vasodilation of rat pial artery in response to hypotension. Am J Physiol 276:H376H3821999

30

Jia YLi PDziennis SWang RK: Responses of peripheral blood flow to acute hypoxia and hyperoxia as measured by optical microangiography. PLoS ONE 6:e268022011

31

Jia YLi PWang RK: Optical microangiography provides an ability to monitor responses of cerebral microcirculation to hypoxia and hyperoxia in mice. J Biomed Opt 16:0960192011

32

Jia YQin JZhi ZWang RK: Ultrahigh sensitive optical microangiography reveals depth-resolved microcirculation and its longitudinal response to prolonged ischemic event within skeletal muscles in mice. J Biomed Opt 16:0860042011

33

Kondo SNajm IKunieda TPerryman SYacubova KLüders HO: Electroencephalographic characterization of an adult rat model of radiation-induced cortical dysplasia. Epilepsia 42:122112272001

34

Kuhl DEEngel J JrPhelps MESelin C: Epileptic patterns of local cerebral metabolism and perfusion in humans determined by emission computed tomography of 18FDG and 13NH3. Ann Neurol 8:3483601980

35

Lado FAMoshé SL: How do seizures stop?. Epilepsia 49:165116642008

36

Lee SKLee SYKim KKHong KSLee DSChung CK: Surgical outcome and prognostic factors of cryptogenic neocortical epilepsy. Ann Neurol 58:5255322005

37

Lee YMessing ASu MBrenner M: GFAP promoter elements required for region-specific and astrocyte-specific expression. Glia 56:4814932008

38

Li TLan JQFredholm BBSimon RPBoison D: Adenosine dysfunction in astrogliosis: cause for seizure generation?. Neuron Glia Biol 3:3533662007

39

Li TLytle NLan JQSandau USBoison D: Local disruption of glial adenosine homeostasis in mice associates with focal electrographic seizures: a first step in epileptogenesis?. Glia 60:83952012

40

Li TRen GLusardi TWilz ALan JQIwasato T: Adenosine kinase is a target for the prediction and prevention of epileptogenesis in mice. J Clin Invest 118:5715822008

41

Li TSteinbeck JALusardi TKoch PLan JQWilz A: Suppression of kindling epileptogenesis by adenosine releasing stem cell-derived brain implants. Brain 130:127612882007

42

Martin KA: Microcircuits in visual cortex. Curr Opin Neurobiol 12:4184252002

43

Masino SALi TTheofilas PSandau USRuskin DNFredholm BB: A ketogenic diet suppresses seizures in mice through adenosine A1 receptors. J Clin Invest 121:267926832011

44

Michel CMMurray MMLantz GGonzalez SSpinelli LGrave de Peralta R: EEG source imaging. Clin Neurophysiol 115:219522222004

45

Najm IMTilelli CQOghlakian R: Pathophysiological mechanisms of focal cortical dysplasia: a critical review of human tissue studies and animal models. Epilepsia 48:Suppl 221322007

46

Neirinckx RD: Evaluation of regional cerebral blood flow with 99mTc-d, 1 HM-PAO and SPECT. Neurosurg Rev 10:1811841987

47

O'Regan MEBrown JK: Abnormalities in cardiac and respiratory function observed during seizures in childhood. Dev Med Child Neurol 47:492005

48

Oghlakian ROTilelli CQHiremath GKAlexopoulos AVNajm IM: Single injection of a low dose of pentylenetetrazole leads to epileptogenesis in an animal model of cortical dysplasia. Epilepsia 50:8018102009

49

Pelligrino DAVetri FXu HL: Purinergic mechanisms in gliovascular coupling. Semin Cell Dev Biol 22:2292362011

50

Phillis JW: Adenosine and adenine nucleotides as regulators of cerebral blood flow: roles of acidosis, cell swelling, and KATP channels. Crit Rev Neurobiol 16:2372702004

51

Phillis JWLungu CLBarbu DEO'Regan MH: Adenosine's role in hypercapnia-evoked cerebral vasodilation in the rat. Neurosci Lett 365:692004

52

Pignataro GMaysami SStuder FEWilz ASimon RPBoison D: Downregulation of hippocampal adenosine kinase after focal ischemia as potential endogenous neuroprotective mechanism. J Cereb Blood Flow Metab 28:17232008

53

Reif RQin JAn LZhi ZDziennis SWang R: Quantifying optical microangiography images obtained from a spectral domain optical coherence tomography system. Int J Biomed Imaging 2012:5097832012

54

Schuele SULüders HO: Intractable epilepsy: management and therapeutic alternatives. Lancet Neurol 7:5145242008

55

Schwartz THChen LMFriedman RMSpencer DDRoe AW: Intraoperative optical imaging of human face cortical topography: a case study. Neuroreport 15:152715312004

56

Sciotti VMVan Wylen DG: Increases in interstitial adenosine and cerebral blood flow with inhibition of adenosine kinase and adenosine deaminase. J Cereb Blood Flow Metab 13:2012071993

57

Shen HYLusardi TAWilliams-Karnesky RLLan JQPoulsen DJBoison D: Adenosine kinase determines the degree of brain injury after ischemic stroke in mice. J Cereb Blood Flow Metab 31:164816592011

58

Siegel AMJobst BCThadani VMRhodes CHLewis PJRoberts DW: Medically intractable, localization-related epilepsy with normal MRI: presurgical evaluation and surgical outcome in 43 patients. Epilepsia 42:8838882001

59

Studer FEFedele DEMarowsky ASchwerdel CWernli KVogt K: Shift of adenosine kinase expression from neurons to astrocytes during postnatal development suggests dual functionality of the enzyme. Neuroscience 142:1251372006

60

Theofilas PBrar SStewart KAShen HYSandau USPoulsen DJ: Adenosine kinase as a target for therapeutic antisense strategies in epilepsy. Epilepsia 52:5896012011

61

Wang RK: Three-dimensional optical micro-angiography maps directional blood perfusion deep within microcirculation tissue beds in vivo. Phys Med Biol 52:N531N5372007

62

Wang RKHurst S: Mapping of cerebro-vascular blood perfusion in mice with skin and skull intact by Optical Micro-AngioGraphy at 1.3 mum wavelength. Opt Express 15:11402114122007

63

Wang RKJacques SLMa ZHurst SHanson SRGruber A: Three dimensional optical angiography. Opt Express 15:408340972007

64

Weinberg MSMcCown TJ: Current prospects and challenges for epilepsy gene therapy. Exp Neurol 244:27352013

65

Weinberg MSSamulski RJMcCown TJ: Adeno-associated virus (AAV) gene therapy for neurological disease. Neuropharmacology 69:82882013

66

Wykes RCHeeroma JHMantoan LZheng KMacDonald DCDeisseroth K: Optogenetic and potassium channel gene therapy in a rodent model of focal neocortical epilepsy. Sci Transl Med 4:161ra1522012

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