RESEARCH ARTICLE
Effect of diagnostic ultrasound during the fetal period on learning and memory in mice

https://doi.org/10.1016/j.aanat.2007.04.008Get rights and content

Summary

Background

An experiment was conducted to find out whether in utero exposure to diagnostic ultrasound leads to changes in postnatal behavior in adult mice.

Methods

A total of 15 pregnant Swiss albino mice were exposed to diagnostic levels of ultrasound (3.5 MHz, 65 mW/cm2, intensity(spatial peak−temporal peak) (ISPTP)=1 mW/cm2, intensity(spatial average−temporal average) (ISATA)=240 mW/cm2) for 30 min on day 14 or 16 of gestation. All exposed as well as control animals were left to complete gestation and parturition. Their offspring were used in our further studies. They were monitored during early postnatal life for standard developmental markers (such as pinna detachment, eye opening and fur development) and postnatal mortality was recorded up to 6 weeks of age. The litters were subjected to behavioral tests for learning and memory at 4 months of age. Representative animals from each group were sacrificed and the hippocampal region of the brain was assayed for biogenic amines, noradrenaline, dopamine, serotonin (5-HT) and 5-HT's metabolite, 5-hydroxy indoleacetic acid (5-HIAA), in order to determine whether ultrasound exposure produced any biochemical changes in the hippocampal region of the brain. Coronal sections from the dorsal hippocampus from the representative animals from each group were processed for staining and the number of neurons was counted.

Results

Neither the standard developmental markers (such as pinna detachment, eye opening and fur development) nor the postnatal mortality was affected by ultrasound exposure. However, there was a significant impairment in learning (hole board test) and memory functions (shuttle box test) in both the exposure groups. Significant reductions in the biogenic amines and the decrease in the neuronal density were found only in day 14th pc ultrasound-exposed group compared with the control animals. The 16th day exposure group is relatively resistant to ultrasound-induced impairment of brain functions.

Conclusions

The results suggest that the early fetal brain is highly susceptible to induction of neurobehavioral changes by ultrasound exposure.

Introduction

The effects of prenatal exposure to diagnostic ultrasound on postnatal behavior have become an important research area, because it was observed that no conclusions were drawn from investigations concerning the actual safety of clinical ultrasound, even though several human epidemiological studies strongly suggest the biosafety of prenatal diagnostic ultrasound exposure (Stark et al., 1984; Smith, 1984; Kinnier-Wilson and Waterhouse, 1984; Bakketeig et al., 1984; Meire, 1987; Ziskin and Petitti, 1988; Maulik, 1989; Brent et al., 1991). In addition to developments in clinical practice, the use of diagnostic ultrasound devices with increasing acoustic output intensities and the more frequent use of ultrasound for preconception and early gestational examination have prompted a reappraisal of possible reproductive risks (Miller, 1991; Martin et al., 1991; AIUM, 1993; Tarantal and O’Brien, 1994).

Experimental efforts to delineate the bioeffects of ultrasound on in utero development have been inconclusive. Some studies have reported increased malformation rates (Saravazayan et al., 1982; Takabayashi et al., 1985), while others have found no such effects (Kimmel et al., 1989; Child et al., 1984, Child et al., 1989; Vorhees et al., 1991a). Furthermore, there are conflicting reports concerning the effects of ultrasound exposure on fetal body weight and behavioral changes. Tarantal and Hendrickx, 1989a, Tarantal and Hendrickx, 1989b observed a slight decrease in body weight and transient behavioral changes in Cynomolgus macaques exposed to 10 min of ultrasound of 7.5 MHz on 21–35 and 36–60 days of gestation. Hande and Uma Devi, 1992, Hande and Uma Devi, 1993, Hande and Uma Devi, 1995, reported a significant decrease in body weight and impaired locomotory and learning performance when mice were exposed to 10 min ultrasound on day 14.5 of gestation. Norton et al. (1991) also reported slower performance in the behavioral test in rats exposed to ultrasound on day 15 of gestation. Vorhees et al. (1991) did not find any changes in the fetal body weight, nor any evidence of embryotoxicity in S.D. rats exposed to 3.0 MHz ultrasound on 4–19 days of gestation. The same authors in 1994 observed effects at the highest intensity on locomotor activity and on measures of errors of commission and time spent finding the goal in a multiple-T water maze, when ultrasound was given for 10 min/day on gestation days 4–20. Suresh et al., 1996, Suresh et al., 2002, Suresh et al., 2006, reported a significant reduction in body weight and a marked effect on locomotory, and learning and memory behavior in mice exposed to ultrasound for 10, 20 and 30 min on gestation days 10–18. Stewart et al. (1985) and Fisher et al. (1996) did not find any visible acute effects of ultrasound.

The hippocampus with its precise morphology plays an important role in learning and memory behavior. Any damage to the hippocampus results in various behavioral alterations (Sienkiewicz et al., 1992, Sienkiewicz et al., 1994; Baskar and Uma Devi, 2000; Hossain and Uma Devi, 2001). Its biogenic amines are also very important in maintaining brain function such as learning and memory. However, there are no reports to show that changes in hippocampal biogenic amines and neuronal density are related to behavioral changes induced by in utero exposure to diagnostic ultrasound. Preliminary studies from our laboratory have shown that the induction of malformations by ultrasound exposure during organogenesis and fetal period induced behavioral changes in adult mice. The present experiment deals with brain functions in relation to learning and memory, hippocampal biogenic amines and neuronal density in the adult mouse.

Section snippets

Selection of material

Virgin Swiss albino female mice 3 months old were placed with same age group males for mating in the ratio 2 females:1 male overnight and examined the next morning for the presence of vaginal plug, a sign of successful copulation. The females with vaginal plugs were separated and labelled as 0-day pregnant. Maternal vaginal temperature was also measured. In a sham control group, 15 pregnant mice without exposure to ultrasound were used for comparison. All animals exposed to ultrasound and

Biochemical study

After completing all of the behavior tests, animals were anesthetized (CO2 inhalation) and sacrificed by decapitation. The brains, including the olfactory bulb, were rapidly removed and transferred to ice-cold saline. The weight of each individual brain was recorded and the hippocampus was immediately separated and weighed. The hippocampal tissue was homogenized in 0.5 ml of 0.4 N ice-cold perchloric acid containing 0.1% EDTA disodium salt, 0.05% sodium metabisulphite and internal standard

Histological study

The brains from representative groups of animals were removed and processed for the histological studies. The animals were deeply anesthetized with ether and fixed on a dissection board whereupon the chest cavity was opened to expose the heart. Approximately 15 ml of 0.9% of saline was perfused through left the ventricle at the rate of 1 ml/min. This was followed by perfusion with 10% formalin, approximately 250 ml/adult mouse, at the same flow rate. Twenty-four hours postfixation the animal was

Statistical analysis

Statistical evaluation was performed using Student's t-test to observe changes in the levels of biogenic amines and the number of neurons of the hippocampus. One-way analysis of variance (ANOVA) for the hole board test, and the Mann–Whitney U-test for the shuttle box test, p<0.05 was considered statistically significant.

Results

Ultrasound exposure for 30 min on day 14 or 16 of gestation raised the maternal vaginal temperature slightly above normal (mean±SE is 0.83±0.14 on day 14 pc and 1.1±0.12 on day 16 pc exposure group). Ultrasound exposure did not produce any change in the developmental markers (such as pinna detachment, eye opening and fur development) of the offspring. Postnatal mortality also was not significantly different from that of the control group (data not shown).

Discussion

These results demonstrate that exposure of the fetal mouse to ultrasound at 14 days of gestation can significantly impair brain function, even though postnatal growth and physiological responses may not be adversely affected.

In the present experiment, it has been found that although the maternal vaginal temperature increased by ∼1.1 °C above normal during ultrasound exposure for 30 min, it did not exceed 39 °C, and dropped immediately after exposure was terminated. However, earlier reports have

References (51)

  • M.C. Ziskin et al.

    Epidemiology of human exposure to ultrasound. A critical review

    Ultrasound Med. Biol.

    (1988)
  • Bioeffects and Safety of Diagnostic Ultrasound

    (1993)
  • L.S. Bakketeig et al.

    A randomized controlled trial of ultrasonographic screening in pregnancy

    Lancet

    (1984)
  • S.B. Barnett et al.

    Identification of mechanism responsible for fetal weight reduction in mice following ultrasound exposure

    Ultrasonics

    (1990)
  • R.L. Brent et al.

    Medical sonography. Reproductive effects and risks

    Teratology

    (1991)
  • S.Z. Child et al.

    A test for the effects of low-temporal average intensity pulsed ultrasound on the rat foetus

    Exp. Cell. Biol.

    (1984)
  • M.J. Durcan et al.

    Time course of ethanols effects on locomotor activity, exploration and anxiety in mice

    Psychopharmacology

    (1988)
  • J.E. Fisher et al.

    Behavioral effects of prenatal exposure to pulsed-wave ultrasound in unanesthetized rats

    Teratology

    (1996)
  • M.P. Hande et al.

    Effects of prenatal exposure to diagnostic ultrasound on the development of mice

    Radiat. Res.

    (1992)
  • M.P. Hande et al.

    Effect of in utero exposure to diagnostic ultrasound on postnatal survival and growth of mouse

    Teratology

    (1993)
  • M. Hossain et al.

    Effect of irradiation at the early foetal stage on adult brain function of mouse: learning and memory

    Int. J. Radiat. Biol.

    (2001)
  • Developmental Effects of Irradiation on the Brain of the Embryo and Fetus

    (1986)
  • R.P. Jensh et al.

    Studies of the effect of 0.4 and 0.6 Gy prenatal X-irradiation on postnatal and adult behavior in the Wister rat

    Teratology

    (1987)
  • J. Jones et al.

    Further neurochemical studies on adult rat following ultrasound exposure during the third trimester of gestation

    J. Ultrasound Med.

    (1988)
  • C.A. Kimmel et al.

    Development exposure of mice to pulsed ultrasound

    Teratology

    (1989)
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