Cytomegalovirus infection of the fetal brain: intake of aspirin during pregnancy blunts neurodevelopmental pathogenesis in the offspring

Fig. 1. Dysregulated fatty acid oxidation pathways and overactivation of the arachidonic acid-to-PG pathway in CMV-infected brains. A Oxygenated fatty acids derived from linoleic acid (HODE) and arachidonic acid (HETE, PgE2 and TxB2) analyzed by mass spectrometry in the present study. B, C Brain content of the different oxygenated fatty acids of interest (derived from linoleic or arachidonic acid). Only data for lipids showing significant dysregulation in CMV-infected brains at E17 (B) and at P1 (C) are shown (see Figure S3 for all other lipids); a statistical trend obtained with the ratio of 13- to 9-HODE at E17 is also indicated. MEM: non-infected brains (E17: n = 8, 1 litter; P1, n = 9, 3 litters); CMV: untreated, CMV-infected brains (E17: n = 12, 1 litter; P1: n = 8, 2 litters); CMV + ASA: ASA-treated, CMV-infected brains (E17: n = 11, 1 litter; P1: n = 8, 2 litters). ****: p < 0.0001; **: p < 0.01; *: p < 0.05; ns: not significant. Kruskal–Wallis test with Dunn's post-hoc correction

Fig. 2. Maternal intake of aspirin reverts the increased microglial expression of Cox-1 in CMV-infected fetal brains. Cox-1 and Cox-2 microglial expressions were quantified in brains from non-infected (MEM) cohort and in brains from untreated (CMV) and ASA-treated (CMV + ASA) infected cohorts, all from two litters each, using immunohistochemistry. A Cells were quantified in a region of interest (ROI; red square) located in the dorsolateral corner of the lateral ventricle, in brain sections selected at the same anatomical level (plate 30–57; see http://larrywswanson.com/?page_id=936) [40]. B Representative confocal images (coronal sections) of embryonic brains at E17 after immunohistochemical stainings for Iba1+ cells (microglia; magenta) and for Cox-1+ (cyan, left panels) and Cox-2+ (cyan, right panels) cells in the three experimental conditions. Scale bars apply to all corresponding images and insets (dashed squares). Left and right insets are labeled with one and two stars, respectively. White arrowheads point to co-labeled Iba1+, Cox-1+ or Iba1+, Cox-2+ cells. Orange arrows point to Iba1+, Cox1− or Iba1+, Cox2− cells. (C) (top) Densities of microglia (Iba1+ cells) were calculated as…

Fig. 3. Maternal aspirin intake does not rescue Cox-1 and Cox-2 microglial overexpressions in CMV-infected neonatal brains. Cox-1 and Cox-2 microglial expressions were quantified in brains from non-infected (MEM) cohort (three litters) and in brains from untreated (CMV) (four litters) and ASA-treated (CMV + ASA) (three litters) infected cohorts, using immunohistochemistry. A Cells were quantified in a region of interest (ROI; red square) located in the dorsolateral corner of the lateral ventricle, in brain sections selected at the same anatomical level (bregma + 0.8 mm; see https://www.inmed.fr/en/en-atlas-stereotaxique-du-cerveau-de-rat-au-cours-du-developpement-postnatal) [41]. B Representative confocal images (coronal sections) of embryonic brains at P1 after immunohistochemical stainings for Iba1+ cells (microglia; magenta) and for Cox-1+ (cyan, left panels) and Cox-2+ (cyan, right panels) cells in the three experimental conditions. Scale bars apply to all corresponding images and insets (dashed squares). Left and right insets are labeled with one and two stars, respectively. White arrowheads point to co-labeled Iba1+, Cox-1+ or Iba1+, Cox-2+ cells. Orange arrows point to Iba1+,…

Fig. 4. Maternal aspirin improves the in vivo postnatal phenotypes after CMV infection of the fetal brain. Postnatal phenotyping was done as reported previously [21] on a daily basis during the two first postnatal weeks by comparing cohorts of rat pups previously submitted to icv injections of MEM (vehicle) or of CMV at E15. CMV cohorts were untreated or treated with either of acetylsalicylic acid (ASA) or N-acetyl-L-cysteine (NAC) provided to the dam in drinking water ad libitum from E5-6 until delivery. MEM: non-infected cohort (n = 30 from three litters); CMV: CMV-infected, untreated cohort (n = 41 from five litters); CMV + ASA: CMV-infected, ASA-treated cohort (n = 55 from six litters); CMV + NAC: CMV-infected, NAC-treated cohort (n = 47 from six litters). Sex ratio did not differ significantly between the four conditions at birth (p = 0.7497, Fisher's exact test). As reported [21], CMV infection worsened the postnatal phenotypes, compared to the MEM cohort. Both the CMV + ASA and the CMV + NAC cohorts displayed significant improvements in: A survival; B body weight gain; C success to the righting reflex test; D success to the cliff aversion test; E occurrence of hindlimb…

Fig. 5. Cox-1 knock-out does not improve the in vivo postnatal phenotypes after CMV infection of the fetal brain. Postnatal phenotyping was done as reported previously [21] on a daily basis during the two first postnatal weeks in 17 litters with Cox-1+/+ (wild-type, WT; n = 35), Cox-1± (heterozygotes, HET; n = 67) and Cox-1−/− (homozygotes knock-out, KO; n = 23) rat pups previously submitted to icv injections of CMV at E15. Sex ratio did not differ significantly between the three conditions at birth (p = 0.2489, Fisher's exact test). No significant improvement was observed in Cox-1−/− and in Cox-1± pups compared to Cox-1 WT pups for A survival B body weight gain C success to the righting reflex test D success to the cliff aversion test E occurrence of hindlimb hyperextension F generalized tonic–clonic epileptic seizures. Of note, success to the righting reflex test even worsened significantly (p = 0.0223) in Cox-1−/− rats compared to Cox-1 WT. Kaplan–Meier method followed by Log-Rank test (A); mixed model for repeated measures (B–F)

Fig. 6. Maternal aspirin intake prevents against ictal- and interictal-like events in CMV-infected postnatal brains ex vivo. A Representative local field potential (LFP) trace of spontaneous events recorded at P15 within the neocortical layer IV in brain slices from CMV-infected rat pups (top). The ictal-like event is shown at an expanded timescale (bottom). B Mean frequency distribution of spikes for ictal-like events (n = 7 events in brain slices from five CMV-infected pups (two litters)). Bin size was set at 5 Hz. Average count values corresponding to the middles of each bin are connected with a solid line. A zoom-in of the initial part (0–45 Hz) of the frequency distribution is shown on the right corner. C Proportion of pups exhibiting ictal-like events in different conditions: control (MEM); untreated, CMV-infected (CMV); and ASA-treated, CMV-infected (CMV + ASA). *: p < 0.05. Fisher’s exact test, two-tailed, with pairwise comparisons and Benjamini–Hochberg correction. D Representative LFP traces of spontaneous interictal-like events recorded in brain slices from control (MEM, top trace), untreated CMV-infected (CMV, middle trace), and ASA-treated CMV-infected (CMV + ASA, bottom…

Fig. 7. Cox-1 knock-out prevents against ictal- and interictal-like events in CMV-infected postnatal brains ex vivo. A Representative LFP trace of spontaneous events recorded at P15 within the neocortical layer IV in brain slices from wild-type (WT) rat pups infected with CMV (top). The ictal-like event is shown at an expanded timescale (bottom). B Mean frequency distribution of spikes for ictal-like events (n = 6 events in brain slices from eight CMV-infected WT pups (four litters)). Bin size was set at 5 Hz. Average count values corresponding to the middles of each bin are connected with a solid line. A zoom-in of the initial part (0–45 Hz) of the frequency distribution is shown on the right corner. C Proportion of pups exhibiting ictal-like events in different groups: CMV-infected, WT pups (WT CMV); CMV-infected, Cox-1 KO pups (KO CMV). **: p < 0.01. Fisher’s exact test, two-tailed. D Representative LFP traces of spontaneous interictal-like events recorded in brain slices from CMV-infected WT (top trace) and CMV-infected Cox-1 KO (bottom trace) pups. E Mean frequency distribution of spikes for interictal-like events. CMV-infected WT (WT CMV): light blue trace, n = 12 slices from six…