Lasting blood-brain barrier disruption induces epileptic focus in the rat somatosensory cortex

Abstract

Perturbations in the integrity of the blood-brain barrier have been reported in both humans and animals under numerous pathological conditions. Although the blood-brain barrier prevents the penetration of many blood constituents into the brain extracellular space, the effect of such perturbations on the brain function and their roles in the pathogenesis of cortical diseases are unknown. In this study we established a model for focal disruption of the blood-brain barrier in the rat cortex by direct application of bile salts. Exposure of the cerebral cortex in vivo to bile salts resulted in long-lasting extravasation of serum albumin to the brain extracellular space and was associated with a prominent activation of astrocytes with no inflammatory response or marked cell loss. Using electrophysiological recordings in brain slices we found that a focus of epileptiform discharges developed within 4-7 d after treatment and could be recorded up to 49 d postoperatively in >60% of slices from treated animals but only rarely (10%) in sham-operated controls. Epileptiform activity involved both glutamatergic and GABAergic neurotransmission. Epileptiform activity was also induced by direct cortical application of native serum, denatured serum, or albumin-containing solution. In contrast, perfusion with serum-adapted electrolyte solution did not induce abnormal activity, thereby suggesting that the exposure of the serum-devoid brain environment to serum proteins underlies epileptogenesis in the blood-brain barrier-disrupted cortex. Although many neuropathologies entail a compromised blood-brain barrier, this is the first direct evidence that it may have a role in the pathogenesis of focal cortical epilepsy, a common neurological disease.

Figure 1.

Bile salt-induced long-lasting BBB opening. a, b, Rats were injected intraperitoneally with the albumin-binding dye Evans blue; pictures show brains 1 hr (a) and 7 d (b) after focal cortical perfusion with DHC. At both time intervals, extravasation of Evans blue-albumin complexes into the parenchyma within the treated area indicates an open BBB. c, d, Under fluorescence, microscopic extravasation of the Evans blue-albumin complex is seen (red) in the treated cortex, but not in the cortex from the sham-operated contralateral window. e, Quantitative image analyses of sections from the left medial (sham-operated), right lateral (not-treated), and right medial (treated) somatosensory cortex (n = 50 sections from each region), show enhanced staining in the treated region. f, Photometric analysis of Evans blue-albumin content in control and treated tissue. Enhanced absorbance was measured at 1 hr and 6 d after treatment. ^*p < 0.05; ^**p < 0.001 (in comparison to control values).

Figure 2.

Bile salts do not induce neuronal toxicity or epileptiform activity in vitro. a, Intracellular recording from a layer V neuron in response to injected current pulses under aCSF (control) and 40 min under 3 mm DHC. b, Simultaneous extracellular (top trace) and intracellular (bottom trace) recordings from the same neuron as in a, in response to white matter stimulation at increasing intensities. c, I-V curve before and after wash-in of DHC. The input resistance was unaltered. d, Quantitative analysis of the field potential integral in seven slices at different stimulus intensities and different concentrations of DHC. Responses were normalized to control values for each slice.

Figure 3.

Electrophysiological responses in BBB-disrupted cortex. a, Electrophysiological recordings were performed in slices from BBB-treated as well as nontreated cortical regions from operated and sham-operated animals. In the BBB-treated region the typical potential evoked in response to white matter stimulation (St₁) showed an early, short-duration potential followed by a late, long-duration, event with a variable latency. Stimulation in the white matter outside the treated region (St₂) often elicited delayed paroxysmal activity limited to the treated region. b, Paroxysmal events were first recorded in slices 4 d after treatment. Scaling refers to a and b. c, Overlapping traces in response to three consecutive extracellular stimuli (0.1 Hz); extracellular (bottom) and intracellular recordings (top) show that synaptic paroxysmal responses are evoked synchronously. Interelectrode distance was ∼300 μm. d, e, The integral of the early (d, <50 msec after stimulus artifact, values for each slice were normalized to the maximal response) and late (e, 50-500 msec) evoked responses in slices from treated (filled symbols) and sham-operated (open symbols) cortices under increasing stimulus intensity (normalized to threshold intensity: I_threshold). T, Treated; S, sham-operated; N, nonoperated.

Figure 5.

Paroxysmal activity is associated with GABA-mediated inhibition: a, Simultaneous intracellular (top traces) and extracellular recordings (Fp, bottom traces) are shown in response to extracellular stimulation. Responses to consecutive stimulation are shown at different membrane voltages in relation to the resting potential (V_r = -70 mV). b, Simultaneous extracellular recordings from the nontreated and treated somatosensory cortex of the same slice. Superimposed traces are shown under aCSF (CON) and 40 min after the addition of bicuculline (BIC). Note the enhanced negative deflection in the field potential recorded in the treated region of the slice. c, Recordings from a slice with spontaneous interictal-like epileptiform activity during perfusion of bicuculline-containing aCSF. Note the initial suppression followed by increase in frequency of appearance and amplitude of spontaneous activity. d, Extracellular recordings during bicuculline perfusion were associated with an initial reduction in duration and amplitude of paroxysmal activity (20′) followed by the appearance of large field potential (40′). e, Quantitative analyses of the integral of early and late evoked responses under bicuculline (n = 4; see Results). ^*p < 0.05 (in comparison to control values).

Figure 4.

AMPA-KA or NMDA receptor antagonists blocked paroxysmal responses. a, b, Slices showing paroxysmal activity under aCSF (CON) were perfused with 30 μm of CNQX to block the AMPA-KA receptors. The abolition of late paroxysmal events (middle trace and black bars) and the significant reduction of early potentials (white bars) are shown by measuring the integral of the maximal response. This effect was partially reversible. c, d, NMDA receptors were blocked by APV (30 μm). Although early PSPs were not affected, late paroxysmal activity was significantly reduced. This effect was fully reversible. ^*p < 0.05 (in comparison to control values).

Figure 6.

Mechanisms underlying BBB-induced epileptiform activity. a, Somatosensory cortex was perfused with aCSF (sham), bile salts (BS), serum, denatured serum (D-Serum), electrolytic solution with serum concentrations (aSerum) and serum albumin in increasing concentrations (Albumin). Representative traces from extracellular recordings 7 d after treatment are displayed. b, Quantification of the averaged integral of the late evoked responses in all slices (white bars) and percentage of slices in which paroxysmal activity was observed (gray bars). Albumin concentrations (100% represents serum levels) are noted. n refers to the number of slices examined under each experimental condition. c, Focal perfusion of albumin-Evans blue solution resulted in penetration of the albumin into the cortex of the ipsilateral (treated) hemisphere. In a different experiment, after intraperitoneal injection of Evans blue, extravasation of fluorescent albumin was more prominent in the DOC-treated compared with the albumin-treated hemisphere. The white bars represent averaged color intensity values (n = 20 sections in each experiment). d, Photometric quantification of Evans blue-albumin brain concentrations in nontreated (control), sham-operated, albumin and DOC-treated brains. Note that only DOC treatment caused a significant increase in dye penetration. Inset, Brain after bilateral cranial window operation and intraperitoneal Evans blue injection. The right hemisphere was perfused with albumin and the left with DOC. ^*p < 0.05; ^**p < 0.01.

Figure 7.

Focal cortical perfusion with DHC or albumin caused astrocytic activation with no neuronal loss. a, b, Whereas histological sections 24 hr after treatment show slight extracellular edema in the treated hemisphere, cortical structure is otherwise normal. Arrow points to the treated, exposed cortical region. Bar graphs represent cell count from histological sections 2 and 14 d (n = 6 each) after treatment in percentages compared with the contralateral nontreated region. c, d, Coronal sections 48 hr after perfusion with albumin, showing normal cortical structure under low magnification. The black boxes represent the cortical regions seen under a larger magnification in d. e, In a large magnification normal cortical structure is observed. Note a typical cortical capillary showing blood cells limited to the vessellumen. f, For comparison, 24 hr after a traumatic cortical injury extravasation of blood cells is observed in the cortical tissue (for methodological details, see Eyupoglu et al., 2003). g, h, A prominent increase in the number of GFAP-stained astrocytes is seen in an albumin-treated cortex compared with the nontreated contralateral cortex. Bar graphs represent counted GFAP-positive cells (in percentage to the contralateral hemisphere) from histological sections 2 and 14 d (n = 6 each) after treatment.