Inflammatory Molecule Profiles in Preschool Children with Neurodevelopmental Disorders Secondary to Perinatal Risks

The study addresses how neurodevelopmental dysfunctions in preschool children—arising from perinatal risks such as term perinatal asphyxia—relate to long-term profiles of inflammatory mediators. Prior work by the group showed that the magnitude of perinatal risks and neurological signs correlates with pro-inflammatory serum patterns, suggesting persistent low-grade inflammation (parainflammation). Here, the authors hypothesize that different expressions of neurological disorders (caregiver-perceived regulatory disturbances, clinically diagnosed dysautonomia and asymmetries, and cognitive impairments) exhibit distinct inflammatory profiles, potentially including opposing patterns, and aim to determine whether a common or divergent inflammatory signature exists across these conditions.

Background literature cited links perinatal complications to neurodevelopmental sequelae and implicates neuroimmune interactions in persistent dysfunction and parainflammation. Cytokines such as IFN-γ, IL-1β, IL-6, IL-8, IL-10, IL-17A, and TNF are highlighted as critical to brain development and function. Prior findings by the authors associated the severity of perinatal damage with pro-inflammatory serum profiles in preschoolers. Broader literature discusses how chronic dysfunction may sustain low-grade inflammation without adequate pro-resolving responses, and that early-life stress and adverse environments can sensitize neuroimmune responses and elevate pro-inflammatory cytokines, with implications for cognition and mental health.

Design: Observational, descriptive, cross-sectional, partially retrospective study in preschoolers born in Mexico City (March 2014–December 2016) with longitudinal neurodevelopmental follow-up. The retrospective component captured dysautonomia diagnosed in the first year of life. Cohort and follow-up: Children were part of the Neurodevelopmental Research Center cohorts (Instituto Nacional de Pediatría). Neurodevelopmental check-ups: monthly <6 months, every 2 months up to 18 months, then every 4 months. The results correspond to the last evaluation (mean age 34.7 months), with inflammatory molecule sampling within one month of that assessment. Two blood samples per protocol (7-month interval). Participants: Term infants (37–42 weeks), eutrophic (mean birth weight 3,245 g); 15 had perinatal asphyxia (Apgar <7 at 1 min and required invasive ventilation); 12 had Apgar >7 and no invasive ventilation. From 45 with neurodevelopmental assessments, 27 had complete inflammatory data. For cognitive analyses, 7 without Bayley III scores were excluded (n=20 for cognition). Inclusion: Age 4–5 years, >3 neurodevelopmental assessments, active in follow-up cohorts for perinatal risks (asphyxia, congenital hypothyroidism, mixed encephalopathy, congenital toxoplasmosis, congenital heart disease), informed consent. Exclusion: Genetic syndromes, cerebral palsy, convulsive syndrome, chronic inflammatory diseases (e.g., cancer, asthma, allergies), missing blood samples or incomplete data. Assessments: - Regulatory risk: DeGangi instrument completed by caregivers, yielding risk across nine domains (self-regulation, attention, sleep, eating, dressing/bathing/touch, movement, hearing/language/sound, vision, attachment/emotional). Total scores divided into tertiles: low, medium, high risk. - Neurological exam by specialists: muscle tone type and severity; reflex maturation/alteration; dysautonomic signs; developmental patterns (sitting, displacement); pyramidal activity organization; detection of asymmetries and dysautonomia. - Developmental testing: Bayley Scales of Infant and Toddler Development III (Bayley-III) across five domains; cognitive domain used for analyses with significant differences. Biospecimen collection: 5–10 ml peripheral blood; serum aliquoted and stored at −70°C. Pre-sampling instructions: avoid chocolate/coffee in prior 24 h; light breakfast (milk and half fruit portion) ≥4 h before sampling; postpone sampling 14 days after any acute illness. Laboratory analyses: - Multiplex (Luminex MAGPIX) using Affymetrix Human Inflammation 20-plex (eBioscience): GM-CSF, IFN-γ, TNF-α, IL-12p70, IL-17A, IL-1β, IL-6, sICAM-1, E-selectin, P-selectin, IFN-α, IL-1α, IL-8, IP-10, MCP-1, MIP-1α, MIP-1β, IL-4, IL-13, IL-10 (manufacturer-specified detection ranges). - ELISAs: Resolvin D1 (RvD1), Lipoxin A4 (LXA4), Leukotriene B4 (LTB4), Annexin A1 (manufacturer ranges/sensitivities provided). Only statistically significant molecules reported. Statistical analysis: Cytokine concentrations standardized to Z-scores (mean 0, SD 1). No age/sex adjustments (no differences found). Nutrition/anthropometry considered adequate across cohort. Group comparisons via ANOVA with Tukey-Kramer post hoc; when assumptions unmet, Wilcoxon tests used. Significance p<0.05; p 0.05–<0.1 treated as marginal and explored with univariate comparisons. Sample characteristics (Table 1): n=27 with inflammatory data; female 40.7%, male 59.3%; mean child age 34.7±12.7 months. Initial diagnoses: perinatal asphyxia 55.6%, congenital hypothyroidism 14.8%, mixed encephalopathy 18.5%, congenital toxoplasmosis 7.4%, congenital heart disease 3.7%. Regulatory risk: low 37.0%, medium 25.9%, high 37.0%. Dysautonomia present 56.6%. Asymmetry present 29.6%. Cognitive impairment (Bayley-III cognitive) present in 40.0% (of n=20). Ethics approvals and informed consent obtained.

- Cohort and analyzable samples: Of 45 children followed, 27 had complete inflammatory profiles for analysis; cognitive analyses included 20 children with Bayley-III cognitive scores. - Regulatory risk vs inflammatory profile: Across low/medium/high DeGangi risk tertiles, mean Z-scores for cytokines showed an inverse relationship with regulatory risk. High-risk group exhibited the lowest concentrations (negative Z-scores) of both pro-inflammatory (IFN-γ, IL-1β, IL-8) and anti-inflammatory (IL-4, IL-13) cytokines, alongside higher lipoxin (positive Z-scores). Low-risk group showed the opposite (higher cytokines with positive Z-scores; lower lipoxin with negative Z-scores). Medium-risk group showed intermediate cytokine reductions (negative Z-scores), and lipoxin closer to low-risk (negative Z-scores). - Neurological alterations (asymmetry, dysautonomia): Children with asymmetry or dysautonomia had lower cytokine levels (negative Z-scores) than those without these alterations (positive Z-scores). Notably reduced pro-inflammatory cytokines included IL-12p70 (asymmetry) and IFN-γ and TNF (dysautonomia). Anti-inflammatory cytokines IL-4 and IL-13 were reduced in both conditions, with IL-10 additionally reduced in asymmetry. - Cognitive development: Children with below-average cognitive development had higher concentrations (positive Z-scores) of multiple pro-inflammatory mediators (IL-1β, IL-6, IL-8, IL-12p70, IL-17A, MIP-1α, IFN-γ, P-selectin) and also elevated IL-4, compared to cognitively average children (negative Z-scores for these molecules). Lipoxin showed the inverse pattern: lower in cognitively impaired (negative Z-scores) and higher in cognitively normal (positive Z-scores). - Proposed patterns: Findings support two contrasting inflammatory patterns in persistent neurodevelopmental dysfunctions: (a) a sustained low-grade, fluctuating pro-inflammatory response with parallel anti-inflammatory activity but deficient pro-resolving response (observed with cognitive impairment); and (b) a depressed pro- and anti-inflammatory response, in some cases with elevated pro-resolving mediators—termed a hyper-resolution state—associated with regulatory disturbances and motor asymmetries/dysautonomia.

The results indicate that persistent neurodevelopmental dysfunctions are associated with distinct systemic inflammatory signatures. Cognitive impairment aligns with a low-grade pro-inflammatory state coupled with insufficient pro-resolving activity (low lipoxin), consistent with literature associating elevated pro-inflammatory cytokines with depressive phenotypes and cognitive stress. In contrast, children with regulatory disturbances, dysautonomia, or motor asymmetries exhibit broadly reduced pro- and anti-inflammatory cytokines, sometimes alongside increased pro-resolving mediators (lipoxin), suggesting a compensatory hyper-resolution response to chronic functional stress. The authors propose a dynamic model where acute challenges evoke typical pro-/anti-inflammatory and resolving phases, but persistent dysfunction drives either sustained low-grade inflammation with poor resolution or a dampened inflammatory tone with heightened resolution mediators. These contrasting profiles underscore heterogeneity in neuroimmune adaptation to chronic functional disorders and support the concept of a detectable immunological "molecular footprint" of dysfunction. Implications include the potential for inflammatory mediators to serve as markers of neurodevelopmental status and guide targeted interventions; early stimulation in the cohort underscores the potential for neuroplastic adaptation, though residual dysfunctions like regulatory disturbances and functional asymmetries may persist and shape inflammatory set-points.

Neurodevelopmental disorders in preschoolers are associated with characteristic, disorder-specific inflammatory profiles. Children with cognitive impairment show a predominance of pro-inflammatory mediators and reduced pro-resolving signals, whereas those with neurovegetative or motor regulatory disturbances display attenuated pro- and anti-inflammatory cytokines with relatively increased pro-resolving mediators. High regulatory risk is associated with a strong pro-resolving (hyper-resolution) state, possibly reflecting adaptation to sustained inflammatory stimuli. These findings suggest a molecular fingerprint of chronic dysfunction and motivate mapping immune/resolution pathways to improve monitoring and targeted management. Future work should validate findings in larger cohorts, longitudinally track trajectories, and employ gold-standard lipidomics to refine pro-resolving mediator profiling.

- Sample size and completeness: Of 45 children, only 27 had complete inflammatory data; cognitive analyses included 20, limiting statistical power and subgroup analyses. - Methodological limitation: Lipid mediators were quantified by ELISA rather than the gold standard of liquid chromatography–mass spectrometry (LC–MS), potentially affecting measurement precision and specificity. - Cross-sectional design with partial retrospective components limits causal inference; timing differences between assessments and biomarker sampling were minimized to ≤1 month but remain a potential source of variability.