Altered Amniotic Fluid Levels of Hyaluronic Acid in Fetal Rats with Myelomeningocele: Understanding Spinal Cord Injury

Abstract

Myelomeningocele (MMC) is a devastating congenital neural tube defect that results in the exposure of spinal cord to the intrauterine environment, leading to secondary spinal cord injury and severe impairment. Although the mechanisms underlying the secondary pathogenesis are clinically relevant, the exact cause of in utero-acquired spinal cord damage remains unclear. The objective of this study was to determine whether the hyaluronic acid (HA) concentration in amniotic fluid (AF) in the retinoic acid-induced model of MMC is different from that in normal controls and whether these differences could have an impact on the viscosity of AF. Our data shows that the concentration of HA in AF samples from fetuses with MMC (MMC-AF) and normal control samples (Norm-AF) were not significantly different at embryonic day 18 (E18) and E20. Thereafter, the HA concentration significantly increased in Norm-AF but not in MMC-AF. Compared with Norm-AF, the concentration of HA in MMC-AF and the viscosity of MMC-AF were significantly lower at E21. Agarose gel electrophoresis confirmed a significant reduction in the HA level of MMC-AF compared with Norm-AF at E21. No HA-degrading activity was detected in MMC-AF. In summary, we identified a deficiency in the AF level of HA and the viscosity of AF in fetal rats with MMC. These data are discussed in relation to a potential role the reduction in the AF viscosity due to the low level of HA may play in the exacerbating effects of mechanical trauma on spinal cord damage at the MMC lesion site.

Keywords: fetal rats; hyaluronic acid; myelomeningocele; neural tube defects; spinal cord injury.

FIG. 1.

(A-C) Photomicrographs showing external views of retinoic acid–induced myelomeningocele (MMC) defect in fetal rats at embryonic Day 18 (E18), E20, and E21, respectively. (A) At E18, the MMC placode is exposed dorsally and represents the most superficial element of the fetus. (C) The MMC placode demonstrates damage and excessive flattening of dorsal tissue at E21. Arrows indicate the beginning of the exposed spinal cord. (D) Histogram illustrates hyaluronic acid (HA) concentrations in MMC-amniotic fluid (AF) and normal control (Norm)-AF samples at different stages during gestation. No significant differences in concentrations of HA were found between MMC-AF and Norm-AF samples at E18 and E20. The concentration of HA in MMC-AF was significantly lower compared with Norm-MMC-AF at E21. Data presented as mean ± standard deviation of 18 randomly selected MMC-AF and Norm-AF samples at E18 and E20, 66 Norm-AF samples, and 92 MMC-AF samples at E21. ***p < 0.001.

FIG. 2.

(A) Representative myelomeningocele (MMC)-amniotic fluid (AF) and normal control (Norm)-AF samples electrophoresed on agarose gel and stained with Stains-all show molecular weight (MW) distribution of hyaluronic acid (HA). Lane1, Mega HA ladder and Hi HA ladder; lanes 2 and 5 represent MMC-AF samples at E21; lanes 4 and 5 represent Norm-AF samples at E21; lane 6, Norm-AF sample digested with Streptomyces hyaluronidase (HYAL); lane 7, high MW HA sample generated by streptococcal bacteria (Sigma-Aldrich), and lane 8, high MW HA sample digested with Streptomyces hyaluronidase. Results are representative of three independent experiments. (B) Relative density of HA smears from MMC-AF and Norm-AF reflects the intensity of respective HA-bound dye. Densitometries were performed using ImageJ Software. The relative density is expressed as the percent of the total amount of HA-bound dye. Data presented as mean ± standard deviation of 12 randomly selected samples. ***p < 0.001.

FIG. 3.

(A) Histogram illustrates hyaluronic acid (HA) concentration in myelomeningocele (MMC)-amniotic fluid (AF) samples at embryonic Day 21 (E21) and after an overnight incubation of MMC-AF with 200 μg/mL of exogenous high molecular weight (HMW) HA used as a substrate or with 200 μg/mL of exogenous HMW HA and Streptomyces hyaluronidase (80 U/mL) used as a positive control. Data presented as mean ± standard deviation of three independent experiments involving six randomly selected MMC-AF samples. ***p < 0.001. (B) Representative analysis of HA staining intensity in MMC-AF samples incubated with HMW HA overnight and control samples electrophoresed on agarose gel and stained with Stains-all. Lane1, Mega HA ladder and Hi HA ladder; lane 2, MMC-AF; lane 3, MMC-AF after an overnight incubation with HMW HA; lane 4, MMC-AF after an overnight incubation with HMW HA and Streptomyces hyaluronidase (HYAL). Results are representative of three independent experiments.

FIG. 4.

(A) Histogram illustrates significantly lower viscosity of myelomeningocele (MMC)-amniotic fluid (AF) compared with normal control samples (Norm)-AF at embryonic day 21 (E21). Data presented as mean ± standard deviation of 14 randomly selected MMC-AF and Norm-AF samples. ***p < 0.001. (B) No significant differences in the total AF protein concentration were found between MMC-AF and Norm-AF at E21. Data presented as mean ± standard deviation of 12 randomly selected MMC-AF and Norm-AF samples.

FIG. 5.

Proposed mechanism contributing to in utero spinal cord damage and abrasion of cells from the exposed spinal cord in myelomeningocele (MMC) fetuses. The layer of amniotic fluid surrounding the fetus present in the amniotic sac provides protection from external trauma or pressure and allows the free movement as the fetus grows. In fetuses with MMC the exposed spinal cord is vulnerable. Reduced hyaluronic acid level and viscosity of amniotic fluid in MMC may abrogate the ability of amniotic fluid to exert its protective action against mechanical trauma causing disruption of the dorsal spinal cord protruding through the MMC defect and shearing of cells from the dorsal surface of the spinal cord into surrounding amniotic fluid. These phenomena might occur particularly late in gestation, when fetal seize and movement increases.