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

Abstract

Background: Congenital cytomegalovirus (CMV) infections represent one leading cause of human neurodevelopmental disorders. Despite their high prevalence and severity, no satisfactory therapy is available and pathophysiology remains elusive. The pathogenic involvement of immune processes occurring in infected developing brains has been increasingly documented. Here, we have used our previously validated rat model of CMV infection of the fetal brain in utero to test whether the maternal administration of four different drugs with immunomodulatory properties would have an impact on the detrimental postnatal outcome of CMV infection.

Methods: CMV infection of the rat fetal brain was done intracerebroventricularly. Each of the drugs, including acetylsalicylic acid (aspirin, ASA), a classical inhibitor of cyclooxygenases Cox-1 and Cox-2, the two key rate-limiting enzymes of the arachidonic acid-to-prostaglandins (PG) synthesis pathway, was administered to pregnant dams until delivery. ASA was selected for subsequent analyses based on the improvement in postnatal survival. A combination of qRT-PCR, mass spectrometry-based targeted lipidomics, immunohistochemistry experiments, monitoring of neurologic phenotypes and electrophysiological recordings was used to assess the impact of ASA in CMV-infected samples and pups. The postnatal consequences of CMV infection were also analyzed in rats knocked-out (KO) for Cox-1.

Results: Increased PGE2 levels and increased proportions of Cox-1⁺ and Cox-2⁺ microglia were detected in CMV-infected developing brains. Maternal intake of ASA led to decreased proportion of Cox-1⁺ fetal, but not neonatal, microglia, while leaving the proportions of Cox-2⁺ microglia unchanged. Maternal intake of ASA also improved the key postnatal in vivo phenotypes caused by CMV infection and dramatically prevented against the spontaneous epileptiform activity recorded in neocortical slices from CMV-infected pups. In contrast with maternal intake of ASA, Cox-1 KO pups displayed no improvement in the in vivo phenotypes after CMV infection. However, as with ASA administration, the spontaneous epileptiform activity was dramatically inhibited in neocortical slices from CMV-infected, Cox-1 KO pups.

Conclusion: Overall, our data indicate that, in the context of CMV infection of the fetal brain, maternal intake of ASA during pregnancy improved CMV-related neurodevelopmental alterations in the offspring, likely via both Cox-1 dependent and Cox-1 independent mechanisms, and provide proof-of-principle for the use of ASA against the detrimental outcomes of congenital CMV infections.

Keywords: CMV; Cox-1; Cyclooxygenase; Fetal brain; Herpes virus; Lipids.

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 the total number of Iba1 + cells per µm² of brain section. (bottom) The proportions of microglia (Iba1⁺) expressing either of Cox-1 or Cox-2 were quantified by calculating the ratio of Iba1⁺, Cox-1⁺, or Iba1⁺, Cox-2⁺ cells to the total number of Iba1⁺ cells. Cox-1 series: MEM: n = 12; CMV: n = 11; CMV + ASA: n = 11. Cox-2 series: MEM: n = 11; CMV: n = 11; CMV + ASA: n = 12. ****: p < 0.0001; ***: p < 0.001; **: p < 0.01; *: p < 0.05; ns: not significant. Kruskal–Wallis test with Dunn's post-hoc correction

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⁺, Cox1⁻ or Iba1⁺, Cox2⁻ cells. C (top) Densities of microglia (Iba1⁺ cells) were calculated as the total number of Iba1 + cells per µm² of brain section. (bottom) The proportions of microglia (Iba1⁺) expressing either of Cox-1 or Cox-2 were quantified by calculating the ratio of Iba1⁺, Cox-1⁺, or Iba1⁺, Cox-2⁺ cells to the total number of Iba1⁺ cells. Cox-1 series: MEM: n = 11; CMV: n = 12; CMV + ASA: n = 13. Cox-2 series: MEM: n = 12; CMV: n = 12; CMV + ASA: n = 13. ***: p < 0.001; *: p < 0.05; ns: not significant. Kruskal–Wallis test with Dunn's post-hoc correction

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 hyperextension. F ASA, but not NAC, led to a significant delay in the occurrence of GTCS. E–F In addition to the mixed model (left statistics), Fisher's exact test (right statistics) was used to assess the overall risks to hindlimb hyperextension (E) and to GTCS (F) in the MEM, CMV-infected and ASA-treated cohorts (see also Figure S6B). Note that due to the previously reported relationship between survival on the one hand and the neurologic phenotypes on the other hand [21], a misleading effect of apparent improvement with time could be perceived. ****: p < 0.0001,***: p < 0.001; **: p < 0.01; *: p < 0.05; ns: not significant. Kaplan–Meier method followed by Log-Rank test (A); mixed model for repeated measures (B–F); Fisher's exact test, two-tailed, with pairwise comparisons and Benjamini–Hochberg correction (E, F)

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 trace) pups. E Mean frequency distribution of spikes for interictal-like events. Control (MEM, grey trace, n = 12 slices from five pups (two litters)); untreated CMV-infected (CMV, blue trace, n = 9 slices from five pups (two litters)); ASA-treated CMV-infected (CMV + ASA, orange trace, n = 12 slices from six pups (three litters)). A zoom-in of the initial part (0–45 Hz) of the frequency distribution is shown on the right corner. F Summary data for averaged spike frequencies for interictal-like events in control (MEM, left, n = 12 slices from five pups (two litters)), untreated CMV-infected (CMV, middle, n = 9 slices from five pups (two litters)) and ASA-treated CMV-infected (CMV + ASA, right, n = 12 slices from six pups (three litters)) conditions. ***: p < 0.001; *: p < 0.05; ns: not significant. Kruskal–Wallis test with Dunn’s multiple comparisons test

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 pups; CMV-infected Cox-1 KO (KO CMV): dark blue trace, n = 9 slices from six pups; all pups from the same four litters. A zoom-in of the initial part (0–45 Hz) of the frequency distribution is shown on the right corner. F Summary data for averaged spike frequencies for interictal-like events in CMV-infected WT (WT CMV) pups (right, n = 12 slices from six pups) and from CMV-infected Cox-1 KO (KO CMV) pups (left, n = 9 slices from six pups); all pups from the same four litters. ****: p < 0.0001. Mann Whitney test, two-tailed