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. 2008 May;4(2):91-9.
doi: 10.1017/S1740925X09990135. Epub 2009 Aug 13.

Uncoupling of astrogliosis from epileptogenesis in adenosine kinase (ADK) transgenic mice

Tianfu Li  1 , Jing-Quan Lan, Detlev Boison
Affiliations

Uncoupling of astrogliosis from epileptogenesis in adenosine kinase (ADK) transgenic mice

Tianfu Li et al. Neuron Glia Biol. 2008 May.

Abstract

The astrocytic enzyme adenosine kinase (ADK) is a key negative regulator of the brain's endogenous anticonvulsant adenosine. Astrogliosis with concomitant upregulation of ADK is part of the epileptogenic cascade and contributes to seizure generation. To molecularly dissect the respective roles of astrogliosis and ADK-expression for seizure generation, we used a transgenic approach to uncouple ADK-expression from astrogliosis: in Adk-tg mice the endogenous Adk-gene was deleted and replaced by a ubiquitously expressed Adk-transgene with novel ectopic expression in pyramidal neurons, resulting in spontaneous seizures. Here, we followed a unique approach to selectively injure the CA3 of these Adk-tg mice. Using this strategy, we had the opportunity to study astrogliosis and epileptogenesis in the absence of the endogenous astrocytic Adk-gene. After triggering epileptogenesis we demonstrate astrogliosis without upregulation of ADK, but lack of seizures, whereas matching wild-type animals developed astrogliosis with upregulation of ADK and spontaneous recurrent seizures. By uncoupling ADK-expression from astrogliosis, we demonstrate that global expression levels of ADK rather than astrogliosis per se contribute to seizure generation.

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Figures

Figure 1
Figure 1
Spontaneous seizures in Adk-tg mice. A, Part of coronal brain section of an adult Adk-tg mouse stained with diaminobenzidine hydrochloride (DAB) for ADK immunoreactivity. Note strong ADK-immunoreactivity in the CA3 pyramidal cells (asterisk); Scale bar: 150 γm. B, C, Spontaneous seizure recorded from the CA3 area of an Adk-tg mouse. The trace shows the intrahippocampal representation from a bipolar electrode. For quantitative seizure data, please refer to Table 1.
Figure 2
Figure 2
Intraamygdaloid KA –induced SE terminated after 20 min with lorazepam in Adk-tg mice causes wt-like acute injury. Coronal brain sections were prepared 24 h after status epilepticus (SE). A, B, Cresyl violet staining showing acute injury restricted to the ipsilateral CA3 induced by SE terminated after 20 min with lorazepam in Adk-tg mice. C, D, TUNEL positive cells in Adk-tg mice subjected to 20 min of SE. E, F, Cresyl violet staining showing ipsilateral CA3 damage induced by SE terminated after 30 min with lorazepam in wild-type (Wt) mice. G, H, TUNEL positive cells in wt mice 24 h after 30 min of SE. Scale bars: A,B,E,F, 300μm, C,D,G,H, 75μm. For quantitative and statistical analysis, please refer to main text.
Figure 3
Figure 3
Selective ADK-deficiency in the ipsilateral CA3. Coronal brain sections were prepared 24 h after 20 min of status epilepticus (SE) in Adk-tg mice. A, B, G, H, NeuN immunofluorescence staining (green) shows neuronal cell loss that is selective for the ipsilateral CA3. C, D, I, J, ADK immunofluorescence staining (red) shows ADK-deficiency in the ipsilateral CA3 that corresponds to the loss of NeuN expressing neurons. E, F, K, L, GFAP immunofluorescence (green) shows maintenance of astrocytes within the injured CA3. Note that the lack of ADK-immunoreactivity within the injured CA3 is a function of the loss of ADK-expressing neurons and the transient downregulation of ADK as a consequence of acute injury. Scale bars, A-D, 150μm, E-H, 75μm. For quantitative and statistical analysis, please refer to Figure 6.
Figure 4
Figure 4
Prevention of spontaneous seizures in Adk-tg mice. Intra-CA3 EEG recordings, followed by immunohistochemical analysis were performed 24 h after 20 min of SE in Adk-tg mice or 30 min of SE in wild-type mice. A-D, Coronal brain section of Adk-tg mice stained with diaminobenzidine hydrochloride (DAB) for ADK immunoreactivity reveal ADK deficiency that is restricted to the ipsilateral CA3 in Adk-tg mice. E, F, Spontaneous seizures recorded from the CA3 area of Adk-tg mice are restricted to the contralateral hippocampus (E) that is characterized by ADK-overexpressing pyramidal neurons (A,C), whereas the ipsilateral CA3, characterized by lack of ADK-expressing neurons (B,D), is devoid of any seizure activity (F). G-J, ADK immunoreactivity in wild-type mice is largely restricted to the cell bodies of astrocytes. Note downregulation of ADK as an adaptive response to acute injury. K, L, EEG recordings taken from the ipsi- and contralateral CA3 of wild-type mice do not reveal any seizure activity. Scale bars, A,B,G, H, 300μm, C,D,I,J, 75μm. For quantitative and statistical analysis, please refer to Figure 6.
Figure 5
Figure 5
Lack of ADK-upregulation and lack of spontaneous seizures in Adk-tg mice 3 weeks after KA. Adk-tg mice subjected to 20 min of SE or wild-type mice subjected to 30 min of SE were analyzed 3 weeks after KA-injection. A, B, Coronal brain sections of Adk-tg mice stained with Cresyl violet reveal the original IPI (neuronal cell loss) that is restricted to the ipsilateral CA3. C, D, ADK immunoreactivity (red) reveals ADK-deficiency in the ipsilateral CA3. Note the lack of upregulated ADK in the ipsilateral CA3. E, F, GFAP immunoreactivity (green) reveals prominent astrogliosis that is restricted to the ipsilateral CA3. G, H, Electroencephalograms recorded from the CA3. Note the lack of spontaneous seizures in the ipsilateral CA3 despite the presence of prominent astrogliosis. I, J, Coronal brain sections of wild-type mice stained with Cresyl violet demonstrate a similar degree of the original IPI. K, L, ADK immunoreactivity reveals upregulation of (astrocytic) ADK in the ipsilateral CA3. M, N, GFAP immunoreactivity reveals prominent astrogliosis restricted to the ipsilateral CA3. O, P, Electroencephalograms recorded from the CA3 demonstrate seizure activity only within the astrogliotic ADK-overexpressing CA3. Scale bars, A, B, I, J, 300μm, C-F, K-N, 75μm. For quantitative and statistical analysis, please refer to Table 1 and Figure 6.
Figure 6
Figure 6
Quantification of ADK immunodensities and GFAP-expressing cells in the CA3 region. A, Relative density of ADK immunoreactivity was determined on DAB-stained sections by scanning CA3a fields of 300 × 200 μm on 2 sections each from n = 6 animals per treatment group 24 h after KA-injection. Data were normalized to the contralateral hippocampus of KA-injected wt mice. B, Total number of GFAP-positive cells in corresponding CA3a fields of 300 × 200 μm was determined by counting GFAP-positive cells on 2 sections each from n = 6 animals per treatment group. Note the similar degree of astrogliosis (i.e. increase in GFAP positive cells) in wt and in Adk-tg mice 3 weeks after KA-injection. C, Relative density of ADK immunoreactivity normalized to the contralateral hippocampus of KA-injected wt mice 3 weeks after KA-injection. Note a significant increase in ADK immunoreactivity in the ipsilateral CA3 of wild-type mice, while ADK-immunoreactivity in the CA3 of Adk-tg mice was comparable to wild-type control. Data analysis was done by ANOVA; mean ± SD. **P < 0.01; ***P < 0.001.
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