Generation of the Early-Gestational Maternal Immune Activation Mouse Model to Assess Prenatal Inflammation on Neurodevelopment

Summary

The article provides a comprehensive protocol to generate a maternal immune activation (MIA) mouse model to study the effects of early-gestational inflammation on neurodevelopment, including key metrics to ensure reliability and reproducibility.

Abstract

Neurodevelopmental disorders (NDDs) arise from complex interactions between genetic and environmental risk factors, including prenatal immune activation. Maternal immune activation (MIA) in mice is a widely used model to study environmental contributions to NDDs; variability in experimental outcomes limits reproducibility. Here, we present a standardized protocol for inducing MIA during early gestation at embryonic day 9.5 (E9.5) in mice, incorporating multiple quality control metrics to assess immune response and predict pregnancy outcomes. Following administration of polyinosinic:polycytidylic acid (poly(I:C)), maternal serum cytokines are quantified to confirm robust immune activation, allowing an exclusion metric for low responders. Maternal weight trajectories from E0.5 to E12.5 are predictive of litter viability, with dams that produced litter exhibiting characteristic weight-gain patterns distinct from dams that lost a litter. We also observed that the animal vendor significantly influenced maternal immune response and litter viability, while poly(I:C) formulation was not a significant factor. Altogether, the protocol provides strategies to enhance the reproducibility of the early-gestational MIA model and the viability of the offspring by monitoring maternal immune response and weight changes, and by controlling animal vendors, to ultimately study the effect of prenatal immune activation on neurodevelopment.

Introduction

Current genome-wide association studies identified hundreds of risk alleles, chromosomal rearrangements, and copy number variants that contribute to neurodevelopmental disorder (NDD) susceptibility^1,2,3,4,5. However, genetic alterations confer only a small degree of increased risk, and the large copy number variants with stronger disease associations affect only a small percentage of cases⁶. Moreover, human epidemiologic studies reveal that early-life stressors, such as prenatal infl....

Protocol

All animal experiments were conducted in accordance with ethical guidelines at the University of Oklahoma and were approved by the Institutional Animal Care and Use Committee (IACUC). Animals were housed in individually ventilated cages on 1/8-inch cellulose bedding with ad libitum access to food and water, and cage enrichments including a nestlet and a hut. The animal breeding facility is maintained on a 14-hour on, 10-hour off light cycle in accordance with Jackson Laboratory to reflect the photoperiod of late spring, a peak mouse breeding season.

Refer to Table 1 for specific data fields to be collected throughout the pr....

Results

Following administration of poly(I:C) or saline at E9.5, maternal serum cytokines were assessed to evaluate the efficiency and robustness of MIA induction (Figure 2). MIA dams displayed significantly increased interleukin-6 (IL-6) levels relative to CON dams (Figure 2A). Additionally, assessment of IL-6 levels allowed for exclusion of MIA dams that did not meet the predefined threshold of 1,000 pg/mL. Thus, IL-6 quantification serves as a valuable biomarker for .......

Discussion

In this protocol, we established checkpoints to increase the efficiency, robustness, and reproducibility of the early-gestational maternal immune activation (MIA) model, focusing on maternal cytokine responses, weight trajectories, and experimental variables that influence pregnancy outcomes. Our results demonstrate that MIA induces variable maternal immune responses, with IL-6 serving as a reliable biomarker for identifying dams with suboptimal immune activation. Approximately 31% of MIA-treated dams failed to reach the.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Animal weighing scale	Kent Scientific	SCL-4000	For weighing mcie
BD Lo-Dose U-100 Insulin syringes - 28G	Fisher Scientific	14-826-79	For PIC, intraperitoneal injection
C57BL/6J female mice	The Jackson Laboratory	000664	10-12 week old, virgin females
C57BL/6J male mice	The Jackson Laboratory	000664	Breeding males
C57BL/6NCrl female mice	Charles River Laboratories	027	10-12 week old, virgin females
Centrifuge (24-place lab standard)	Eppendorf	5425 R	Spin down blood to collect serum
Data documentation platform	Airtable, Microsoft Excel, etc.		Keep records of important data, such as weights, time of injection, etc. (Table 1)
Digital dry bath - One block	Medicus Health	5277M1	Use to heat PIC before injection
Eppendorf tubes - 1.5 mL	Eppendorf	30123611	Use when diluting PIC to stock nd working concentrations
Fisherbrand gauze sponges	Fisher Scientific	MSD-1400250	Use to stop bleeding of tail after blood collection
Fisherbrand mall probe 6 in.	Fisher Scientific	13-820-026	Use to plug check
HydroGel	Clear H20	70-01-5022	Supplement after injections
Mouse IL-6 uncoated ELISA kit	Thermo Fisher Scientific	88-7064-88	Quantify IL-6 levels in maternal serum
Nalgene polypropylene Griffin low-form plastic beakers	Thermo Fisher Scientific	1201-1000PK	Put mice in beaker when weighing
Poly(I:C) (LMW)	Invivogen	tlrl-picw	PIC (LMW) for MIA injection
Polyinosinic–polycytidylic acid sodium salt	Sigma	P9582	PIC (Sigma) for MIA injection
Scalpel blades, #22, 10 pack	Home Science Tools	DE-SC22BLD	Use to snip end of tail for blood collection
Sodium chloride 0.9% (Normal saline), USP, Sterile grade, Cytiva Hyclone	Cytiva	Z1376	Inject for generation of control mice
Tail snipping platform	Braintree Scientific, Inc.	IL-200	Properly restrains mice during tail bleeds
Vortex-Genie 2	Scientific Industries, Inc.	SI-0236	Vortex PIC after dilution