Name: Modeling Ascending Vaginal Infection, Preterm Birth, and Neonatal Morbidity in Mice (Video) | JoVE Journal
Uploaded: 2025-10-10T12:35:00.000+00:00
Duration: 4 min 18 s
Description: Learn a mouse model for studying preterm birth caused by ascending vaginal bacterial infection using Escherichia coli. This method enables real-time tracking of infection progression and analysis of neonatal outcomes.

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5 minutes

Escherichia coli K1 A192PP-luxABCDE was kindly gifted by Professor Peter Taylor, University College London. Figure 1 and Figure 3 were created in&#160; BioRender.com.&#160;This research was supported by the Wellcome Clinical&#160;Research Career Development Fellowship 222970/Z/21/Z&#160;(N.S.), Action Medical Research and&#160;Borne grant GN2647 (N.S., S.N.W., and&#160;D.P.); and Wellbeing of Women grant RG2365&#160;(A.K.B., N.S., M.H., S.N.W., and D.P.).

The authors have no relevant disclosures.

Here, we describe the induction of a novel mouse model of preterm birth and neonatal morbidity via bacterial vaginal ascension. We chose E. coli K1 as it is a common cause of neonatal sepsis and meningitis and is vertically transmitted from the mother to the fetus38,39,40. Key to the success of this model is the use of Pluronic thermosensitive gel, which prevents the leakage of the bacterial suspension from the vagina, allowing for infection to establish and ascend to the uterus. As this model uses live bacteria derived from a septicemia isolate, it is crucial to monitor the dams closely27. If the animals refuse to move or eat or do not care for their pups (e.g., neglect nesting and feeding), they must be humanely sacrificed immediately.
Mostly, symptoms associated with infection dissipate within 24 h of delivery. It is also essential to be aware that different serotypes of E. coli and other strains of bacteria will have varying effects on gestational length, the extent of infection/inflammation, and neonatal outcome. We suggest performing preliminary dose experiments with different CFUs when using new bacterial serotypes/strains to determine the minimum concentration needed for the desired phenotype and setting strict humane end points to prevent suffering. Extreme caution should be used when delivering a live pathogen; allow the literature to guide the appropriate use of these organisms when modeling ascending infection.
The mouse strain is also important, as their immune systems differ. For example, the timing and CFUs used in this model do not have the same outcomes when performed in C57BL/6 mice. We chose to use the C57BL/6 Tyrc-2J mice as time course experiments showed significantly more infection in this strain compared to CD-1, BALB/c, and C57BL/6. In addition, there is evidence of black fur quenching bioluminescent signals, which is a useful measurable outcome in this model29. The optimal gestational day for bacterial administration may also differ with pathogen and mouse strain. There is huge heterogeneity in preclinical PTB models; therefore, it is important to consider these variables in order to develop reproducible results17.
Another consideration is the length of time the animals are exposed to anesthesia. As excessive exposure during pregnancy is associated with neurotoxicity in pups, it is essential to keep the time spent under anesthesia to a minimum (e.g., maximum 5 min) and to always include controls exposed to the same environment and treated with a vehicle substance41,42,43. Additional control groups useful for this model include untreated, intravaginal delivery of Pluronic gel only, and anesthesia only.
Immunohistochemistry is widely used as an experimental tool to identify different proteins and cells, and it can be used to characterize structure and connectivity in the brain. Here, we describe how to analyze key cells of the central nervous system that are associated with neuroinflammation and neurological injury. We also present methods to assess cellular apoptosis and histological stains that are useful for measuring brain and lung morphology. Important considerations to ensure the success of these protocols begin with the careful use of the PAP pen; if the PAP pen touches the tissue, the staining will fail but if the parameter is too wide, the sections could dry out overnight. Similarly, the humidity chamber must be kept damp to avoid drying out the sections.
When performing the Nissl staining, the glacial acetic acid step is the most critical; the sections need to be checked constantly. When the main structures of the brain become visible, the sections need to be moved immediately into EtOH to stop the reaction. We chose to assess the cortex and the hippocampus, as reduced cortical thickness and reduced hippocampal volume have been reported in human premature neonates and other animal models but other regions could be assessed for morphology changes44,45,46,47. Close attention must also be paid to the development of DAB staining. This should be timed and kept consistent between experiments.
Data analysis is the most common limitation of immunohistochemistry due to the difficulty in quantifying the results, which are often considered to be subjective. While GFAP analysis is performed using computer-assisted optical luminosity measurements, inconsistent staining can affect these measurements. In addition, the brain and lung morphology assessments involve manual, freehand measurements relying on the observer&#39;s judgment. To avoid inconsistency, we repeat these assessments twice and in a short period of time. To further reduce any bias, it is important that the assessor is blinded to the treatment groups. Low immunoreactivity can also be a limiting factor. Lack of signal could suggest that the antigen of interest is absent or it could be due to a protocol failure. To address this, we include a tissue sample that can act as a positive control where possible and repeat the staining procedure when we observe a low or absent signal to ensure this was not the result of an experimental error.
In addition to the protocols described, immunological analyzes of the maternal, fetal, and neonatal tissues can be performed by qPCR and ELISA. We have observed increased inflammatory mRNA and protein levels in animals exposed to infection25,31,48. We hypothesize that the neonatal neuropathology we demonstrate histologically could be the result of fetal brain, lung, and gut inflammation25,31. Other neuropathology assessments we have performed include myelin basic protein (MBP) to investigate myelination and NeuN to stain neuronal nuclei25,31. Further to this, other markers can be assessed for neutrophils and microglia to better understand the phenotype and behavior of these cells following exposure to infection in utero49,50. Additionally, behavioral tests could be performed to determine the long-term impact on offspring.
This model is not without limitations. For example, we see variation in pathology both between and within litters. This could be the result of using a live bacterium, where the spread of infection cannot be controlled. Consequently, the pattern and reach of ascending infection into the uterine horns vary and are often asymmetrical. Another limitation is the pathogenicity of the E. coli, which can sometimes cause the dams to become too unwell to care for their pups. Mice are also not the best model of lung injury, due to alveologenesis taking place postnatally, rather than prenatally, as in humans51. We failed to inflate our lung tissues when the samples were collected, which could have impacted on our results. However, our results are consistent with the changes observed in neonatal BPD animal models52,53.
This model recapitulates the postulated human condition whereby bacterial infection ascends from the vagina into the uterus, stimulating early delivery. Following our initial publication of this model, others have replicated or produced similar models, further emphasizing the reproducibility and relevance of this methodology19,54. Crucially, this model exhibits the common morbidities experienced by premature neonates, where other models have failed. This could be due to the use of a bacterial strain that is specifically associated with neonatal sepsis. LPS, which is most commonly used to induce preterm labor in animal models, is lethal to pups, so neonatal outcomes cannot be measured18. Other models of ascending infection have also struggled with producing surviving pups or in demonstrating neonatal morbidity19,20,21,22,23,24,55,56. Clinically relevant animal models, like the one we describe, are fundamental for understanding the pathophysiology of preterm birth and for developing innovative interventions that will improve neonatal outcomes.

Preterm birth (PTB; delivery &#60; 37 weeks) is the main contributor to neonatal mortality worldwide1,2. Accounting for 11% of all births, it is a major global health problem with a complex etiology that is poorly understood, making it difficult to predict and prevent3,4,5. Neonatal survivors of PTB may experience adverse neurodevelopmental outcomes, including cerebral palsy, respiratory disorders, such as bronchopulmonary dysplasia (BPD), and gastrointestinal disturbances such as necrotizing enterocolitis (NEC)6,7,8. Therefore, there is an urgent need for clinically relevant, reproducible animal models to address the biological pathways contributing to PTB and develop innovative interventions.
There are a number of possible mechanisms underlying spontaneous PTB, such as decidual senescence, a breakdown of maternal-fetal tolerance, stress, short cervical length, or cervical damage9. However, at least 40% of spontaneous PTBs are reported to be associated with microbial infection10,11. Microbes&#160;ascending from the vaginal canal through the cervix&#160;to the uterus are&#160;hypothesized and widely accepted&#160;as a common route of infection due to the correlation&#160;between the microbes isolated from the amniotic fluid and those resident in the vaginal microbiota12,13,14. Microorganisms often associated with preterm delivery include species of Mycoplasma, Ureaplasma, Fusobacterium, and Streptococcus, and most cases are polymicrobial15,16.
Mice are often used to model PTB, with the most common approach being the administration of the bacterial toxin lipopolysaccharide (LPS), which can be delivered via intrauterine, intraperitoneal, or intra-amniotic routes to induce levels of inflammation necessary to initiate parturition17. However, LPS does not model live microbial infection, and importantly, neonatal outcomes cannot be measured in these models as LPS induces fetal death18. In recent years, animal models of ascending vaginal bacterial infection have been developed but recapitulating both preterm birth and the associated neonatal outcomes has been a challenge19,20,21,22,23,24.
Our group has established a novel model of ascending vaginal bacterial infection, preterm birth, and neonatal morbidity that recapitulates the pathophysiology of human PTB25. Here, we demonstrate how to assess neonatal neuropathology and lung injury in pups exposed to maternal ascending vaginal infection with a pathogenic Escherichia coli (E. coli) strain that has been linked to human neonatal meningitis. This model offers a unique opportunity to explore the pathophysiology of ascending infection, as well as providing the means to evaluate the effectiveness of preterm labor interventions in improving neonatal outcomes.

<table><tbody><tr><td>Anti-GFAP, rabbit</td><td>Agilent Technologies</td><td>Z033429-2</td><td></td></tr><tr><td>Anti-IBA1, rabbit&amp;nbsp;</td><td>Wako</td><td>019-19741</td><td></td></tr><tr><td>Anti-Ly6G, mouse</td><td>BioLegend</td><td>127601</td><td></td></tr><tr><td>AVT-Horn 184 3CCD camera</td><td>Sony</td><td></td><td></td></tr><tr><td>Biotin-16-DUTP</td><td>Merck</td><td>11093070910</td><td></td></tr><tr><td>Borosilicate Glass Rectangular Coverslips</td><td>FisherScientific</td><td>12363128</td><td></td></tr><tr><td>Cresyl Violet acetate</td><td>ThermoFisher Scientific</td><td>405760100</td><td></td></tr><tr><td>Cryostat CM1900</td><td>Leica</td><td></td><td></td></tr><tr><td>Glass cuvette&amp;nbsp;</td><td>Merck</td><td>BR472700</td><td></td></tr><tr><td>Diaminobenzidine (DAB)</td><td>Generon</td><td>40480004-3</td><td></td></tr><tr><td>Dissecting Forceps, Stainless Steel, 10 cm, Serrated, Curved</td><td>World Precision Instruments Ltd</td><td>15915</td><td></td></tr><tr><td>Dissecting Forceps, Stainless Steel, 10 cm, Serrated, Straight</td><td>World Precision Instruments Ltd</td><td>15914</td><td></td></tr><tr><td>Dissecting Scissors, 10 cm long, Straight</td><td>World Precision Instruments Ltd</td><td>14393</td><td></td></tr><tr><td>DPX&amp;nbsp;</td><td>Merck</td><td>100579</td><td></td></tr><tr><td>Eosin</td><td>Sigma-Aldrich</td><td>HT110116</td><td></td></tr><tr><td>Formaldehyde solution 4% buffered</td><td>Merck</td><td>100496</td><td></td></tr><tr><td>Goat Anti-Rabbit IgG Antibody (H+L), Biotinylated</td><td>Vector Laboratories</td><td>BP-9100-50</td><td></td></tr><tr><td>Goat Anti-Rat IgG Antibody (H+L), Biotinylated</td><td>Vector Laboratories</td><td>BA-9400-1.5</td><td></td></tr><tr><td>GraphPad Prism v.8&amp;nbsp;</td><td>GraphPad Software</td><td></td><td></td></tr><tr><td>H2O2</td><td>Sigma-Aldrich</td><td>7722-84-1</td><td></td></tr><tr><td>Hematoxylin</td><td>Sigma-Aldrich</td><td>MHS16</td><td></td></tr><tr><td>ImageJ</td><td>National Institutes of Health</td><td></td><td></td></tr><tr><td>Isoflurane (IsoFlo)</td><td>Zoetis</td><td>50019100</td><td></td></tr><tr><td>IVIS Lumina II In Vivo Imaging System/Living Image Software</td><td>Perkin Elmer/Revvity</td><td></td><td></td></tr><tr><td>Microslides</td><td>VWR</td><td>ISO8037/I</td><td></td></tr><tr><td>Optimas 6.51&amp;nbsp;</td><td>Media Cybernetics Inc.</td><td></td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>158127</td><td></td></tr><tr><td>Phosphate-buffered saline (PBS)&amp;nbsp;</td><td>Thermo Fisher Scientific (Life Technologies)</td><td>14190094</td><td></td></tr><tr><td>Pluronic F-127&amp;nbsp;</td><td>Sigma-Aldrich</td><td>P2443</td><td></td></tr><tr><td>Quinine</td><td>Scientific Laboratory Supplies</td><td>Q0132</td><td></td></tr><tr><td>ReadyProbes Hydrophobic Barrier Pap Pen</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>R3777</td><td></td></tr><tr><td>Sodium hydroxide</td><td>Honeywell</td><td>S8045</td><td></td></tr><tr><td>Sodium Phosphate Monobasic Anhydrous</td><td>FisherScientific</td><td>BP329-1</td><td></td></tr><tr><td>Sucrose</td><td>Merck</td><td>S9378</td><td></td></tr><tr><td>Terminal transferase&amp;nbsp;</td><td>Merck</td><td>3333574001</td><td></td></tr><tr><td>VECTASTAIN ABC-HRP Kit, Peroxidase</td><td>Vector Laboratories</td><td>PK-4000</td><td></td></tr><tr><td>Xylenes (histological grade)&amp;nbsp;</td><td>Sigma-Aldrich</td><td>534056</td><td></td></tr></tbody></table>

modeling ascending vaginal infection, preterm birth, and neonatal morbidity in mice

Modeling Ascending Vaginal Infection, Preterm Birth, and Neonatal Morbidity in Mice

Preterm birth continues to be a primary cause of neonatal mortality and morbidity globally, with limited preventative therapies. There is an imperative need for clinically 
relevant and reproducible animal models for the translation of innovative interventions 
to become a reality. Various mouse models of preterm birth exist. However, they do not recapitulate human neonatal morbidities, including neuropathological changes and 
lung injury, which are characteristic sequelae of ascending infection and preterm birth. We have characterized a model which, to our knowledge, is the only one that mimics the human scenario whereby these neonatal morbidities are&#160;present following the induction of preterm birth by a live bacterium that ascends from the vagina. This paper demonstrates the&#160;intravaginal administration of a bioluminescent strain of Escherichia coli and details the assessment of neuropathological outcomes and lung injury using immunohistochemistry and histology techniques.

This protocol presents a mouse model of ascending vaginal infection and describes methods for assessing neonatal neuropathology and respiratory outcomes. The procedure to induce ascending vaginal infection should be rapid (within 5 min) and mice should recover within a couple of minutes. Dams developed symptoms of infection after approximately 24 h25. This includes slow movement, piloerection, and a hunched posture. The approximate average delivery time in the infected mice was 40.3 h after E. coli administration (PBS control group mean 52.7 h), occurring on E18.5 compared to term delivery of the PBS control group on E19.531. The percentage of live born pups per litter was reduced (from 100% to 71.4%) with infection, as was their survival over 7 days postnatally (Figure 5)25. When bioluminescent bacteria are used, imaging can track the spread of infection in dams and pups. Bacteria were observed in the uterus, fetal membranes, placenta, and in some fetuses within 18 h of administration (Figure 6), and inflammatory mediators, measured by qPCR and ELISA, were upregulated in these tissues, as well as in the perinatal pup tissues, such as the brain, lungs, and gut25,29,31.
A battery of neuropathology assessments, such as assessing morphology, cell death, microglial activation and astrocyte activation, was performed on the fetuses and neonates using histology and immunohistochemistry25,31,33. Lung tissue can also be evaluated (Figure 4 and Figure 7). Positive staining for TUNEL suggests DNA degradation, a later stage of cell death34. We see a significant increase in TUNEL staining in the cortex, pyriform cortex, external capsule, hippocampus and the thalamus in the brains of pups exposed to E. coli infection during pregnancy (Figure 8A,B)31. IBA-1-labeled microglia, the resident immune cells of the brain, can be categorized by their morphological state, whereby ramification suggests a resting phenotype, while activated and phagocytic cells become amoeboid or rounded in shape35. Therefore, rather than quantifying microglial cells, we present a strategy to assess their phenotype as an indication of their state. We see an increase in IBA1+ ramification in the external cortex and striatum of E. coli pups, compared to the PBS group (Figure 8C,D)31. GFAP-labeled reactive astrocytes, which can be associated with inflammation and neurological injury, were increased in the pyriform cortex and the external capsule in E. coli pups (Figure 8E,F)31,36,37. Nissl staining allows for the assessment of gross morphological changes to the structure of the brain; Cresyl violet is a standard histological stain for neurons and labels both extra nuclear RNA granules and cell nuclei. In the E. coli group, we see reduced thickness of the cortex (Figure 9)31. Similarly, H&amp;E, which respectively stain the nucleus and cytoplasm of cells, allows for assessment of lung morphology (Figure 4)31. We see increased airspaces in E. coli exposed mice, as well as an increase in the number of Ly6G+ neutrophils, an indication of inflammation (Figure 7)31.
For data analyses, time to delivery was analyzed by an unpaired t-test if the data were normally distributed. For the percentage data for live-born pups, an arcsine square root transformation was first required to transform a proportion distribution (bounded by the range 0 to 1) to a normal distribution, which is one of the requirements to be able to perform a t-test. Pup survival was analyzed by a Log-rank (Mantel-Cox) test. For histology and immunohistochemistry, either Mann-Whitney U test or multiple t tests with Welsh's correction were used, depending on the type of data and whether they were distributed normally. For sufficient statistical power, we recommend using a minimum of five dams per treatment group.
<img alt="Mouse experiment process diagram: E. coli intravaginal administration, timeline post-exposure studies." class="xfigimg" src="/files/ftp_upload/66723/66723fig1.jpg" data-altupdatedat="1783396841000" data-alt="Figure 1"> 
Figure 1: Intravaginal administration of Escherichia coli and the treatment timeline to induce preterm birth and neonatal morbidity in mice. (A) E. coli [1], followed by 20% Pluronic gel F127 [2], is administered into the mouse vagina using a sterile 200 µL pipette tip. Quinine powder is added to the introitus [3]. (B) E. coli is delivered into vagina on embryonic day 16.5. Tissues, such as the lungs and brain, can be collected during the fetal or neonatal period. Abbreviation: EN = embryonic day N; PN = postnatal day N; PBS = phosphate-buffered saline. <a href="https://www.jove.com/files/ftp_upload/66723/66723fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Histology brain section diagram labeling cortex, hippocampus, thalamus; anatomical study." class="xfigimg" src="/files/ftp_upload/66723/66723fig2.jpg" data-altupdatedat="1783396841000" data-alt="Figure 2"> 
Figure 2: Brain regions assessed for neuropathology. An example of Nissl staining of a neonatal pup brain and the regions that could be assessed for neuropathology. All brain regions can be assessed for TUNEL, IBA-1, and GFAP. We chose to assess the cortex and hippocampus morphology using Nissl. Magnification: 1x. Scale bar = 1.5 mm. Abbreviations: TUNEL = terminal deoxynucleotidyl transferase dUTP nick end labeling; IBA-1 = allograft inflammatory factor 1; GFAP = glial fibrillary acidic protein. <a href="https://www.jove.com/files/ftp_upload/66723/66723fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Neuron network diagram; shows cell body and branches layout for neurobiology study." class="xfigimg" src="/files/ftp_upload/66723/66723fig3.jpg" data-altupdatedat="1783396841000" data-alt="Figure 3"> 
Figure 3: Positive IBA-1 staining and ramification index assessment. To quantify the ramification index of IBA-1+ cells, count the number of cell bodies within the grid and the average number of branches crossing the horizontal and vertical gridlines. Abbreviation: IBA-1 = allograft inflammatory factor 1. <a href="https://www.jove.com/files/ftp_upload/66723/66723fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Lung tissue analysis; histology images, graph; airspace comparison, PBS vs. E. coli experiment." class="xfigimg" src="/files/ftp_upload/66723/66723fig4.jpg" data-altupdatedat="1783396841000" data-alt="Figure 4"> 
Figure 4: Airspace measurements in pup lungs (H&amp;E staining). (A) Outline the airspaces and measure using the freehand tool in ImageJ. (B) Average airspace area is increased in pups exposed to E. coli compared to PBS. (C) Representative H&amp;E images of pup lungs exposed to PBS and E. coli in utero. Magnification: 40x. Scale bar = 59.5 µm. Abbreviations: H = hematoxylin; E = eosin. n = 2 from 4 litters. *p &lt; 0.05. This figure was adapted from Boyle et al.31. <a href="https://www.jove.com/files/ftp_upload/66723/66723fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="E. coli effects on delivery time, pup survival; data charts; statistical analysis; biomedical study." class="xfigimg" src="/files/ftp_upload/66723/66723fig5.jpg" data-altupdatedat="1783396841000" data-alt="Figure 5"> 
Figure 5: Effects of intravaginal E. coli on time to delivery and pup survival. (A) Time to delivery is reduced in the presence of E. coli. (B) The percentage of pups born alive is significantly reduced and (C) pup survival is reduced in E. coli-exposed pups. Time to delivery: n = 8-10 litters per group; live born pups: n = 10-17 litters per group; pup survival: n = 10-12 litters per group. *p &lt; 0.05, ***p &lt; 0.001. This figure was adapted from Boyle et al.31. <a href="https://www.jove.com/files/ftp_upload/66723/66723fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Luminescence imaging in mice; fetal tissue analysis; diagram shows radiance over time." class="xfigimg" src="/files/ftp_upload/66723/66723fig6.jpg" data-altupdatedat="1783396841000" data-alt="Figure 6"> 
Figure 6: Bioluminescence imaging to monitor E. coli spread. (A) Bioluminescent E. coli ascends from the vagina into the uterine horns within 48 h. (B) Dissected uterine horns show infection with E. coli within 18 h, (C) as do the placenta and fetal membranes. This figure was adapted from Suff et al.25 and Boyle et al.31. <a href="https://www.jove.com/files/ftp_upload/66723/66723fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Neutrophil infiltration in E. coli vs. PBS, microscopy images, Ly6G+ cell graph data, inflammation analysis." class="xfigimg" src="/files/ftp_upload/66723/66723fig7.jpg" data-altupdatedat="1783396841000" data-alt="Figure 7"> 
Figure 7: Ly6G staining for neutrophils in fetal lungs. (A) Staining for Ly6G (positive: arrowheads), to assess neutrophil influx, in PBS and E. coli exposed fetuses. (B) The average number of Ly6G+ cells is significantly increased in E. coli fetuses. Magnification: 40x. Scale bar = 59.5 µm. n = 10 from 5 litters per group. *p &lt; 0.05. Abbreviations: Ly6G = Lymphocyte antigen 6 complex locus G6D. This figure was adapted from Boyle et al.31. <a href="https://www.jove.com/files/ftp_upload/66723/66723fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="TUNEL assay microscopy and GFAP, IBA1 immunostaining graphs; cell death, ramification analysis." class="xfigimg" src="/files/ftp_upload/66723/66723fig8.jpg" data-altupdatedat="1783396841000" data-alt="Figure 8"> 
Figure 8: Examples of TUNEL, IBA1, and GFAP staining. (A) TUNEL staining for cell death in the postnatal pup brain exposed to PBS or E. coli. (B) The average number of TUNEL+ cells is increased in the cortex, pyriform cortex, external capsule, hippocampus, and thalamus. (C) IBA1 immunohistochemistry for microglia in PBS and E. coli groups. (D) Ramification index, as a measure of microglia activation, is increased in the external capsule and striatum in E. coli pups. (E) GFAP staining for astrocytes, (F) which is increased in the pyriform cortex and external capsule in E. coli pups. Scale bar = 59.5 µm. Abbreviations: GFAP = glial fibrillary acidic protein; TUNEL = terminal deoxynucleotidyl transferase dUTP nick end labeling; Pyr = Pyriform cortex; EC = external capsule; Str = striatum; Hip = hippocampus; thal = thalamus; total = all regions; CA = cornu ammonis; OLV = optical luminosity values. Immunohistochemistry analyses: n = 5-7 from 5 litters per group. *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001, n.s. - non-significant. This figure was adapted from Boyle et al.31. <a href="https://www.jove.com/files/ftp_upload/66723/66723fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Brain tissue staining comparison; diagram and graphs showing PBS vs. E. coli impacts on brain metrics." class="xfigimg" src="/files/ftp_upload/66723/66723fig9.jpg" data-altupdatedat="1783396841000" data-alt="Figure 9"> 
Figure 9: Example of positive and negative Nissl staining and morphology assessments. (A) Nissl staining of the postnatal pup brain to assess tissue structure and morphology. (B) There is no impact on brain width but (C) cortical thickness is reduced in pups exposed to E. coli. (D) There is no difference in hippocampal volume. Magnification: 1x. Scale bar = 1.5 mm. n = 5-7 from 5 litters per group. **p &lt; 0.01. This figure was adapted from Boyle et al.31. <a href="https://www.jove.com/files/ftp_upload/66723/66723fig9large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Primary Antibody</td>
			<td>Dilution</td>
			<td>Secondary Antibody/Dilution</td>
			<td>Dilution</td>
		</tr>
		<tr>
			<td>Rabbit anti-GFAP</td>
			<td>   1:6000 </td>
			<td>Goat anti-rabbit</td>
			<td>   1:100  </td>
		</tr>
		<tr>
			<td>Rabbit anti-IBA1</td>
			<td>   1:1000 </td>
			<td>Goat anti-rat</td>
			<td>   1:100  </td>
		</tr>
		<tr>
			<td>Mouse anti-Ly6G</td>
			<td>   1:1000 </td>
			<td>Goat anti-rabbit</td>
			<td>   1:100  </td>
		</tr>
	</tbody>
</table>
Table 1: Antibodies for immunohistochemistry.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Step</td>
			<td>Reagent</td>
			<td>Time (min)</td>
		</tr>
		<tr>
			<td>1</td>
			<td>NISSL stain</td>
			<td>10</td>
		</tr>
		<tr>
			<td>2</td>
			<td>Fresh Distilled Water</td>
			<td>2</td>
		</tr>
		<tr>
			<td>3</td>
			<td>Fresh Distilled Water</td>
			<td>2</td>
		</tr>
		<tr>
			<td>4</td>
			<td>70% EtOH</td>
			<td>2</td>
		</tr>
		<tr>
			<td>5</td>
			<td>90% EtOH </td>
			<td>2</td>
		</tr>
		<tr>
			<td>6</td>
			<td>96% EtOH</td>
			<td>2</td>
		</tr>
		<tr>
			<td>7</td>
			<td>96% EtOH with 3-5 drops glacial acetic acid</td>
			<td>2-5 (CHECK CONSTANTLY)</td>
		</tr>
		<tr>
			<td>8</td>
			<td>100% EtOH</td>
			<td>2</td>
		</tr>
		<tr>
			<td>9</td>
			<td>Isopropanol</td>
			<td>2</td>
		</tr>
		<tr>
			<td>10</td>
			<td>Xylene</td>
			<td>2</td>
		</tr>
		<tr>
			<td>11</td>
			<td>Xylene</td>
			<td>2</td>
		</tr>
		<tr>
			<td>12</td>
			<td>Xylene</td>
			<td>2</td>
		</tr>
	</tbody>
</table>
Table 2: Nissl procedure.

Preterm birth (delivery &#60; 37 weeks) is an urgent global health issue with suboptimal prevention and treatment options. We present a mouse model of ascending vaginal bacterial infection-induced preterm birth and outline how to analyze the resulting neonatal morbidity and mortality.

Preterm birth continues to be a primary cause of neonatal mortality and morbidity globally, with limited preventative therapies. There is an imperative ...

To begin, add 10 milliliters of antibiotic-treated Luria-Bertani, or LB broth, to a sterile 30-milliliter universal container. Retrieve a frozen glycerol stock of Escherichia coli for minus 80 degrees Celsius storage. Using a sterile pipette tip, scrape the surface of the frozen stock to lightly coat the tip with bacteria, then eject the tip directly into the universal container containing the LB broth.Seal the container with parafilm, label it as a biohazard, and place it in an orbital shaker set at 200 RPM and 37 degrees Celsius overnight. The next morning, dilute the overnight bacterial culture 1 to 100 by adding 0.1 milliliters of the culture to 9.9 milliliters of fresh LB broth. Return the diluted culture to the orbital shaker at 200 RPM and 37 degrees Celsius for 1.5 to three hours.Now, pipette 100 microliters of the culture and duplicate into a clear, flat-bottomed 96-well plate and measure the absorbance using a spectrophotometer with path length correction activated. When the optical density at 600 nanometers reaches between 0.5 and 0.7, remove 100 microliters of the bacterial culture. Dilute this in 900 microliters of sterile PBS.Centrifuge the sample at 14, 000g for one minute to wash the bacterial pellet and remove the broth. Discard the supernatant, resuspend the pellet in sterile PBS, and then dilute the suspension 1 to 10, 000 in sterile PBS to achieve 100 to 1000 colony forming units. After anesthetizing the mouse, administer 20 microliters of mid-logarithmic phase E.coli suspension into the vagina using a sterile 200-microliter pipette tip.Then, using a fresh sterile 200 microliter pipette tip, deliver 20 microliters of 20%Pluronic F-127 gel into the vagina to prevent bacterial leakage. Dab quinine powder onto the introitus using sterile gauze to prevent the mouse from cleaning the area. After launching the imaging software, click on Initialize and wait for the camera to reach the operating temperature indicated by a green and locked temperature bar.After 48 hours, place the animal in the supine position on the heated stage of a bioluminescence imaging machine while maintaining anesthesia. Check the luminescent imaging box. Ensure the excitation filter is set to block and the emission filter is set to open.Then, select auto-exposure time. This figure illustrates the time-dependent progression of bioluminescent E.coli infection from the vaginal tract to uterine and fetal tissues in pregnant mice. Bioluminescent E.coli reached the uterine horns within 48 hours post-infection, with strong luminescence signals detected at embryonic day 18.5, in contrast to the absence of signal at embryonic day 16.5.Dissected uterine horns reveal clear bioluminescence signals as early as 18 hours after infection indicating rapid intrauterine colonization. Infection had also reached fetal compartments within 18 hours, as shown by luminescent signals in the placenta, fetal membranes, and amniotic fluid.

modeling-ascending-vaginal-infection-preterm-birth-neonatal-morbidity

Research

JoVE Journal

Immunology and Infection

Source: Seman, B. G., et al. A Neonatal Imaging Model of Gram-Negative Bacterial Sepsis. J. Vis. Exp. (2020)The video demonstrates a neonatal mouse model for studying systemic infection using encapsulated bioluminescent bacteria. Neonatal pups are injected with high- or low-dose bacterial suspensions and returned to their mother for postnatal care. The encapsulated bacteria evade early immune detection, spread systemically, and are visualized over time using luminescence imaging. This model...

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Human Cytomegalovirus (HCMV) infection of neonates represents an important cause of mental retardation, yet the molecular events leading to virus-induced pathogenesis are still poorly understood. To investigate the dynamics of brain infection, we adapted whole-animal in vivo imaging to perform time-course analysis of neonates infected with a luciferase-recombinant virus.

Use of In vivo Imaging to Monitor the Progression of Experimental Mouse Cytomegalovirus Infection in Neonates

Encephalopathy of prematurity encompasses the central nervous system abnormalities associated with injury from preterm birth. This report describes a clinically relevant rat model of in utero transient systemic hypoxia-ischemia and intra-amniotic lipopolysaccharide administration (LPS) that mimics chorioamnionitis, and the related impact of infectious stimuli and placental underperfusion on CNS development.

Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats

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A Controlled Mouse Model for Neonatal Polymicrobial Sepsis

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Probiotic Studies in Neonatal Mice Using Gavage

We developed a model of chorioamnionitis to simulate fetal exposure to maternal inflammation (FEMI) without complications of live organisms to examine the effects of FEMI on development of the offspring’s intestinal tract. This allows for study of mechanistic causes for development of intestinal injury following chorioamnionitis.

A Murine Model of Fetal Exposure to Maternal Inflammation to Study the Effects of Acute Chorioamnionitis on Newborn Intestinal Development

Infection of neonatal mice with bioluminescent E. coli O1:K1:H7 results in a septic infection with significant pulmonary inflammation and lung pathology. Here, we describe procedures to model and further study neonatal sepsis using longitudinal intravital imaging in parallel with enumeration of systemic bacterial burdens, inflammatory profiling, and lung histopathology.

A Neonatal Imaging Model of Gram-Negative Bacterial Sepsis

The protocol presents a mouse model of vaginal colonization with anaerobically cultured human vaginal bacteria. We focus on Gardnerella vaginalis, while including suggestions for Prevotella bivia and Fusobacterium nucleatum. This protocol can also be used as a guide for vaginal inoculations and viable recovery of other anaerobically grown bacteria.

Models of Murine Vaginal Colonization by Anaerobically Grown Bacteria

This protocol outlines a neonatal mouse model of necrotizing enterocolitis (NEC) and subsequent isolation of lamina propria immune cells from the neonatal murine small intestine.

A Neonatal Mouse Model of Necrotizing Enterocolitis and Lamina Propria Isolation for Immune Cell Profiling

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.

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

Learn a mouse model for studying preterm birth caused by ascending vaginal bacterial infection using Escherichia coli. This method enables real-time tracking of infection progression and analysis of neonatal outcomes.

Modeling Ascending Vaginal Infection, Preterm Birth, and Neonatal Morbidity in Mice

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