Postictal behavioural impairments are due to a severe prolonged hypoperfusion/hypoxia event that is COX-2 dependent

8 figures and 5 tables

Figures

Figure 1 with 1 supplement
Seizures induce severe postictal hypoxia.

(A) Local tissue oxygenation in the hippocampus of an awake, freely-moving rat (blue). Green denotes normoxia while red denotes severe hypoxia. (B) Representative oxygen profile before, during, and …

https://doi.org/10.7554/eLife.19352.003
Figure 1—figure supplement 1
Postictal severe hypoxia generalizes to other seizure models.

Both stimulations resulted in severe hypoxia. A 40 s seizure was elicited with MES stimulation and resulted in 104.1 min of severe hypoxia (red). Following 2 min of 3 Hz stimulation, hippocampal pO2

https://doi.org/10.7554/eLife.19352.004
Figure 2 with 1 supplement
Seizures cause postictal vessel constriction in an in vitro preparation and reduced blood flow (hypoperfusion) in vivo.

(A) Location of implants for simultaneous blood flow and pO2 recordings. These two probes were placed at opposing angles to leave room for cable attachment. (B) The simultaneous measurement of mean …

https://doi.org/10.7554/eLife.19352.005
Figure 2—figure supplement 1
Post-seizure administration of nifedipine prevents severe postictal hypoxia.

(A) Nifedipine (15 mg/kg) was administered immediately after a seizure. No differences were observed in seizure duration, however nifedipine initiated a quicker return to baseline pO2(n = 5). (B) …

https://doi.org/10.7554/eLife.19352.006
Figure 3 with 1 supplement
Representative seizure-specific local postictal hypoperfusion in clinical epilepsy.

(A) ASL-010, 26 year old female with drug resistant focal epilepsy. Ictal EEG recording on a longitudinal bipolar montage with left frontal seizure onset and spread to the right frontal region. …

https://doi.org/10.7554/eLife.19352.009
Figure 3—figure supplement 1
Clinical postictal hypoperfusion is more severe with longer seizures.

(A) Cerebral blood flow from baseline and postictal ASL scans. Each point represents in individual. Group mean ± SEM are expressed in grey. Patient number is displayed on the left of the …

https://doi.org/10.7554/eLife.19352.010
Figure 4 with 1 supplement
COX-2 activity during a seizure is required for postictal severe hypoxia.

(A) COX-2 expression in sagittal section. Hippocampal inset is displayed (see B–D). Scale bar = 2 mm. (B–D) Contents of inset from (A). Many NeuN-expressing neurons (B) in DG and hilus express COX-2 …

https://doi.org/10.7554/eLife.19352.011
Figure 4—figure supplement 1
COX-1 or postictal COX-1/2 inhibition do not inhibit severe hypoxia.

(A) SC-560 (20 mg/kg) pre-administration did not significantly alter seizure duration or the resulting oxygen profile (n = 5). (B) Quantification of (A). No differences in the severe hypoxia were …

https://doi.org/10.7554/eLife.19352.012
Genetic knockdown of COX-2 function prevents postictal severe hypoxia.

(A) COX-2 proteins from wild-type and mutant mice (PTGS2Y385F) displayed as a schematic. Both mice have functional peroxidase (POX). Mutants have a point mutation in the cyclooxygenase (COX) active …

https://doi.org/10.7554/eLife.19352.013
Preventing postictal severe hypoxia prevents behavioral impairments.

(A) Experimental timeline for neocortex experiments. Rats were injected with vehicle or nifedipine 30 min prior to a baseline hang time measurement. Neocortical seizures (or sham) were then elicited …

https://doi.org/10.7554/eLife.19352.016
Author response image 1
Susceptibility of Area Below Baseline analysis to changes in baseline pO2.

(A) Seizure duration is not correlated with total area under curve (R square=0.000013, p=0.99). (B) Instead, the baseline pO2 is strongly correlated with the area below baseline (R square=0.52, ***p<…

https://doi.org/10.7554/eLife.19352.018
Author response image 2
Comparing area below baseline analyses with pre- and post-administration of nifedipine.

(A) Pre-administration of nifedipine had no effect on area below baseline. (B) Post-administration of nifedipine significantly reduced the area below baseline (p<0.01).

https://doi.org/10.7554/eLife.19352.019

Tables

Table 1

Characteristics of patients recruited to ASL study.

https://doi.org/10.7554/eLife.19352.007

Patient

Age

Age at Seizure Onset

Structural MRI findings

ASL-001

38

34

Normal

ASL-003

41

13

Lesion in the right superior temporal gyrus, resolving?

ASL-004

25

5

Normal

ASL-006

33

7

Normal

ASL-007

33

29

Bilateral subependymal heterotopias along lateral ventricle right > left

ASL-008

42

22

Right amygdala enlargement, Right occipital cavernoma

ASL-009

40

9

Normal

ASL-010

26

1

Left frontal malformation of cortical development

ASL-011

22

14

Normal

ASL-012

20

0.5

Postsurgical changes in the right frontal lobe, right anterior temporal lobectomy

Table 2

Concordance of postictal ASL hypoperfusion with brain areas involved in the seizure. EEG localization of ictal activity was determined by an Epileptologist based on scalp EEG. GTC denotes when a …

https://doi.org/10.7554/eLife.19352.008

Patient

EEG localization of seizure [duration (S)]

Area of maximal hypoperfusion for ASL quantification

ROI volume (cm3)

Concordant

ASL-001

Left temporal [71]

Left temporal

2.52

yes

ASL-003

Unclear [26]

No Change

1.45

n/a

ASL-004

Left frontocentral [87]

Left superior posterior temporal

0.31

yes

ASL-006

Left fronotemporal [114]

Left insula, left anterior temporal

1.46

yes

ASL-007

GTC, Left hemisphere. Maximal posterior temporo-parietal [64]

Multifocal bihemispheric

1.35

yes

ASL-008

GTC, Right temporo-occipital [155]

Multifocal bihemispheric

1.63

yes

ASL-009

Left fronto-central [85]

Multifocal bihemispheric

0.76

yes

ASL-010

GTC, Bifrontal, maximum left [166]

Left frontal

3.82

yes

ASL-011

Left fronto-temporal [56]

No Change

1.10

n/a

ASL-012

Bitemporal right>left [30]

Multiple areas over right temporal posterior and superior to resection cavity

2.19

yes

Table 3

Investigation of mechanisms involved in postictal severe hypoxia.

https://doi.org/10.7554/eLife.19352.014

Drug

Principle known mechanism of action

Δ severity of hypoxia(VehDrug)Veh × 100%

Nifedipine (15 mg/kg)

L-type Ca2+ Channel Blocker

+78.82 ± 7.632% ***

Nifedipine (15 mg/kg postictal)

+87.33 ± 10.49% **

Acetaminophen (250 mg/kg)

COX-1/2 Inhibitor

+99.88 ± 0.12% ***

Acetaminophen (150 mg/kg)

+88.88 ± 7.355% ***

Acetaminophen (50 mg/kg)

+48.85 ± 26.61%

Acetaminophen (250 mg/kg postictal)

−3.33 ± 17.29%

Ibuprofen (20 mg/kg)

+99.23 ± 0.396% ***

Celecoxib (20 mg/kg)

COX-2 Inhibitor

+67.26 ± 27.42% *

SC-560 (20 mg/kg)

COX-1 Inhibitor

+19.42 ± 15.37%

Celecoxib (20 mg/kg) + SC-560 (20 mg/kg)

COX-2 and COX-1 Inhibitors

+95.80 ± 4.21% ***

Acetaminophen (250 mg/kg) + Nifedipine (15 mg/kg)

COX-1/2 Inhibitor + L-type Ca2+ Channel Blocker

+100 ± 0.00% ***

CAY-10526 (2 mg/kg)

Prostaglandin E2 Synthesis Inhibitor

+41.72 ± 9.94% **

Seratrodast (10 mg/kg)

Thromboxane A2 Receptor Antagonist

−12.91 ± 44.08%

Ozagrel (10 mg/kg)

Thromboxane A2 Synthesis Inhibitor

+21.24 ± 16.42%

2-APB (3 mg/kg)

IP3r Antagonist + TRP Channel Blocker

+45.27 ± 18.44% *

Chelerythrine Chloride (15 mg/kg)

PKC Inhibitor

+25.54 ± 29.90%

Milrinone (3 mg/kg)

Phosphodiesterase-3 Inhibitor

−10.74 ± 34.31%

Sildenafil (15 mg/kg)

Phosphodiesterase-5 Inhibitor

−19.34 ± 14.03%

SKA-31 (10 mg/kg)

IKCa Channel Activator

−1.50 ± 12.68%

Paxilline (2.5 mg/kg)

BKCa Channel Blocker

+19.55 ± 14.98%

L-Arginine (500 mg/kg)

Nitric Oxide Precursor

−5.05 ± 14.08%

Fasudil (10 mg/kg)

Rho Kinase Inhibitor

−53.07 ± 17.27% *

  1. All drugs were delivered by intraperitoneal injection pre-seizure (unless otherwise stated).

  2. Statistics reported as different from chance (one sample T-test).

  3. *p < 0.05, **p<0.01, ***p<0.001.

  4. + number indicates inhibition of hypoxia.

  5. − number indicates potentiation of hypoxia.

Table 4

Effect of Anti-Seizure drugs on postictal severe hypoxia.

https://doi.org/10.7554/eLife.19352.015

Drug

Principal known mechanism of action

Δ severe hypoxia

(VehDrug)Veh × 100%

Ethosuximide (300 mg/kg)

T-type Ca2+ Channel Blocker

+30.53 ± 19.31% *

Topiramate (50 mg/kg)

Na+ Channel Blocker, GABA Enhancement, AMPA Inhibition

+4.34 ± 14.07%

Bumetanide (2.5 mg/kg)

NKCC1 Transporter Inhibitor

−0.92 ± 14.72%

Phenobarbital (30 mg/kg)

GABA Receptor Agonist

−1.22 ± 13.90%

Levetiracetam (250 mg/kg)

Glutamate Release Inhibition

−14.85 ± 19.71%

Phenytoin (75 mg/kg)

Na+ Channel Blocker

−15.76 ± 17.00%

Lamotrigine (15 mg/kg)

Na+ Channel Blocker

−20.95 ± 17.13%

Valproate (150 mg/kg)

Na+ Channel Blocker, GABA Enhancement

−24.89 ± 22.65%

  1. All drugs were delivered by intraperitoneal injection pre-seizure.

  2. Statistics reported as different from chance (one sample T-test).

  3. *p<0.05.

  4. + number indicates inhibition of hypoxia.

  5. -number indicates potentiation of hypoxia.

Table 5

Stereotaxic coordinates for surgical implantation.

https://doi.org/10.7554/eLife.19352.017

Experiment

Anterior(+)/ Posterior(−)

Lateral

Right(+)/Left(−)

Ventral (from brain surface)

Rat Dorsal Hippocampus

Electrode: −3.0 mm

Optode: −3.5 mm

Electrode: 0.5 mm

Optode: 3.5 mm

Electrode: 3.5 mm

Optode: 3.5 mm

Rat Dorsal Hippocampus with LDF

Electrode: −3.0 mm

Optode: −5.0 mm

LDF probe: −3.0 mm

Electrode: 0.5 mm

Optode: 2.2 mm

LDF probe: 3.5 mm

Electrode: 3.5 mm

Optode: 3.5 mm

LDF probe: 3.5 mm

Rat Ventral Hippocampus

Electrode: −4.5 mm

Optode: −3.0 mm

Electrode: 4.5 mm

Optode: 3.5 mm

Electrode: 6.5 mm

Optode: 3.5 mm

Rat Ventral Hippocampus w/ Cannula

Electrode: −4.5 mm

Optode: −3.0 mm

Cannula: - 5.8 mm

Electrode: 4.5 mm

Optode: 3.5 mm

Cannula: −4.5 mm

Electrode: 6.5 mm

Optode: 3.5 mm

Cannula: 4.3 mm

Rat Neocortex

Electrode:+1.0 mm

Optode: 0 mm

Electrode: 0.5 mm

Optode: 3.0 mm

Electrode: 3.6 mm

Optode: 1.5 mm

Mouse Ventral Hippocampus

Electrode: −2.9 mm

Optode: −1.6 mm

Electrode: 3.0 mm

Optode: 2.0 mm

Electrode: 3.0 mm

Optode: 1.8 mm

Download links