ELSEVIER

Applied Acoustics, Vol. 48, No. 3, pp. 187-193, 1996 Copyright 0 1996 Elsevier Science Ltd

Printed in Great Britain. All tights reserved 0003-682X/96/$15.00+0.00

0003-682X(95)00051-8

Acoustical Damage to the Rat Brain

A. K. Singh, J. Behari

School of Environmental Sciences, Jawaharlal Nehru University, 110 067, New Delhi, India

&

P. Raghunathan

NMR Division, All India Institute of Medical Sciences, New Delhi, India

(Received 19 July 1995; accepted 29 August 1995)

ABSTRACT

It has been concluded from the proton nuclear magnetic resonance spectrum of a sonicated rat brain that a peak recognized as lactate appears at 1.3 ppm; the same peak was absent in a normal brain spectrum. The cause of the appearance of this lactate peak was neuronal degradation: neuronal degra- dation leads to release of lactate. The reasons for the damage could be assigned to acoustical streaming produced by acoustical vibrations (4.0 W at 15.0 kHz). Gases dissolved in soft or hard tissue (skull) form bubbles which oscillate with their resonant frequency causing acoustic streaming and microstreaming. This streaming has produced neuronal damage, and hence the appearance of the lactate peak at 1.3 ppm. Copyright 0 1996 Elsevier Science Ltd

Keywords: Brain, proton nuclear magnetic resonance, acoustical damage, lactate peak.

INTRODUCTION

Acoustic energy is transformed into different forms of energy when exposed to the biological system. The mechanism of interaction can possibly be divi- ded into three major categories comprising a thermal effect, cavitational effect and another mechanism including streaming and microstreaming.

187

188 A. K. Singh, J. Behari. P. Raghunuthun

Exposure to ultrasound can be broadly divided into two classes: from the air and from liquid. Exposure to airborne ultrasound occurs in many industrial applications such as cleaning, emulsifying, welding and flow detection and through the use of consumer devices such as dog whistles, rodent repellents and alarms. Liquid-borne exposure occurs predominantly through medical uses in diagnostic therapy and surgery. This more frequent use of ultrasound has increased the possibility of human exposure, raising concern about possible health effects. Studies on ultrasound biohazards provide possible information on the effects of risk for medical, occupational and general population exposure to ultrasound.

The effects reported in human subjects exposed to airborne ultrasound include temporary threshold shift in sound perception, altered blood-sugar level, electrolyte imbalance, fatigue, headaches, nausea and irritability. It has been observed’ that an ultrasound beam of 0.75 MHz at therapeutical intensities directed towards the heart of living rats resulted in the appearance of free haemoglobin in the plasma. Evidence of intravascular haemolysis in vitro,’ probably due to a cavitational mechanism, after exposure to a 25 kHz tissue disrupter has also been reported. The evidence that ultra- sound exposure of cells in suspension can lead to cell lysis is extensive and unquestionable. Cavitation has been reported as the major cause of cellular disruption.3 It is not clear that ultrasound can provide cell lysis in the absence of cavitation effects. Changes to the extracellular membrane follow- ing ultrasonic irradiation are usually manifested as changes in permeability to ion transport; sublethal alteration in the thermocyte plasma membrane that leads to a decrease in potassium content following 1 W/cm2 irradiation in vitro at 1.8 MHz.~ Electron microscopy of cells following ultrasound treatment at therapy intensities has revealed damage to a variety of organ- elles, primarily to mitochondria. When intact tissues have been studied using this technique damage to lysosomes has been seen with consequent release of lysosomal enzymes. It is not clear if lysosomal damage is a direct or indirect result of ultrasonic exposure.‘.”

Ultrasound exposure apparently alters both cellular ultrastructure and metabolism. Cells exposed to ultrasound appear to be more prone to cell death during mitosis. Suppression of cellular growth has been reported under continuous wave (CW) and or pulsed exposure criteria. If the intensity of ultrasound is sufficient death or some type of anatomical abnormality will result in certain organisms.

It is beyond doubt that ultrasound interacts with biological systems and may produce a health risk or biohazards. The literature gives inconsistent results and, therefore, there is an urgent need to pursue work towards investigation of ultrasound biohazards.

Acoutical damage to the rat brain 189

MATERIAL AND METHODS

We have investigated the effect of a 15.0 kHz CW high-amplitude acoustic signal generated by a magnetostriction ferrite transducer. The input power given to the ferrite transducer was 72 W and the output power was 4 W at the frequency of vibration. The WaveTek-166 produces a sinusoidal wave of 3.0 V amplitude at 15.0 kHz, which was fed to the power amplifier and the amplified signal was then fed to the magnetostriction ferrite transducer. We attempted to study the metabolic damage to a rat brain due to 4.0 W acoustical vibrations. Sound in the audible range exerts pressure on the brain legion. The importance of the work is to study the effect of sound pollution on the metabolic properties of the brain.

Proton nuclear magnetic resonance (NMR) studies have the potential of providing access to a much larger number of metabolites compared to other nuclei because of the greater sensitivity and 100% natural abundance. Hence, it was possible to obtain detailed biochemical information on energy metabolism,’ free amino acids, fatty acids and, and neurotransmitters. This technique has been applied here to study the effect on the brain of Wistar rats.

Proton NMR

Proton NMR is, in principle more than 15 times sensitive than 31P or 13C. There are however, significant problems in its applications. One is the pre- sence of an extremely strong resonance from water, the other is the difficulty of resolving the peaks of interest from the multitude of protonated molecules present in the sample.

Magnetic resonance spectroscopy (MRS) was carried out using a Bruker BIOSPEC 47/40 spectroscope, at an operating frequency of 200 MHz corre- sponding to proton resonance. For every animal three coronal and trans- verse slices with a slice thickness of 2.0 mm were acquired covering the brain area. The image protocol follows the specification of multislice/multiecho (MSME), repetition time (Ta) of 1200 ms and echo time (Tn) 30 ms.

Volume selective spectroscopy-spin echo for monitoring the metabolites was carried out using the same system, with water suppression using the VSEL method. Hermite pulses were used, the spectral width was 5 kHz. voxel size 6 x 6 x 6 mm3, TR = 2 s, TE = 135 ms and an average from I536 readings. In both cases the same specifications were maintained.

RESULTS AND DISCUSSION

NMR images of control and sonicated rat brain are shown in Figs 1 and 2 and the NMR spectrum is shown in Figs 3 and 4. The spectra in the two

190 A. K. Singh, 1. Behari, P. Raghunathan

Fig. 1. Proton NMR image of a normal rat brain of a Wistar rat.

Fig, 2. Proton NMR image of a sonicated rat brain.

Acoutical damage to the rat brain 191

I I I II I I I I I I 7 6 5 4 3 2 I 0 -1 -2

Fig. 3. Proton NMR spectrum in a normal rat brain.

3.5 3.0 2.5 2.0 1.5 I.0 0.5 0 -0.5 -1.0 -1.5

eem

Fig. 4. Proton NMR spectrum in an acoustically irradiated rat brain.

cases were significantly different. The images of the sonicated rat brain were less intense in comparison to the control (Fig. l), but we found it very diffi- cult to conclude anything from these images. Hence, more studies are needed to arrive at a detailed conclusion. This could be, at the best, taken as an impetus to carry out an in-depth investigation into how acoustic signals affect functioning of the brain at a molecular level.

We then realized that MRS might be more revealing. These spectra are shown in Figs 3 and 4.

192 A. K. Singh, J. Behuri, P. Raghunathan

From Figs 3 and 4 we noticed a water peak appearing at 4.8 ppm, this was the most prominent one, and a localized shim enabled a line width of 23 Hz, allowing suppression of the water signal. This enabled the metabolite peaks to appear. In a normally developed rat brain, proton metabolite peaks appear at different levels: 2.0, 1.5, 1.3 and 1.0 ppm. N-acetyl aspartate (NAA) appeared at about 2.0 ppm and other metabolites occurred at higher peaks (see Fig. 4). Two small peaks at 1 .O and 1.5 ppm could be assigned to protons of low molecular phospholipids.’

The sonicated rat brain (15.0 kHz vibration at 4.0 W) showed entirely different metabolic activities (cf. Fig. 4) and a highly noisy picture appears in this case. It is expected that acoustical exposure of this intensity has produced various stresses within the brain system, resulting in changes in the relative positions of the intracellular organelles and break-up in the normal structure. The cause of the fall of the intensity in the sonicated brain may be due to altered membrane permeability. It was further noticed that the cellular ultrastructure and metabolism both were altered in the sonicated rat brain.

The prominent metabolic peak appearing at 2.0 and 3 ppm in normal rat brain also appears at the same scale in the case of sonicated rat brain. But the most remarkable observation from these NMR spectra was the appearance of a lactate peak at 1.3 ppm, which did not appear in the normal case. This may be due to neuronal degradation, since neuronal degradation leads to the release of lactate,* which is of a very low concentration in normal rat brain. The cause of damage could be assigned to acoustical streaming. This streaming had produced neuronal damage, and hence the appearance of the lactate peak at 1.3 ppm.

It is beyond doubt that rat brain was affected temporarily. The major cause of damage to the rat brain was acoustical streaming produced by acoustical vibrations. Gases dissolved in a soft or hard tissue (skull) form bubbles which oscillate with their resonant frequency causing acoustic streaming and microstreaming.

REFERENCES

1. Wong, Y. S. & Watmough, D. J, Haemolysis of red blood cells in vitro and in vitro by ultrasound at 0.75 MHz and at therapeutic intensity levels. In Ultra- sound Interactions in Biology and Medicine, ed. R. Millner, E. Rosenfeld and U. Cobet, pp. 179984, Plenum Press, New York, 1983.

2. Ghater, B. V. & Williams, A. R., Absence of of platelet damage following the exposure of non-turbulent blood to therapeutic ultrasound. Ultrasound Med. and Biol., 8 (1982) 85-87.

3. Martin, C. J., Gregory. D. W. & Hodgkiss, M., The effects of ultrasound in vitro on mouse liver in contact with an aqueous coupling medium. Ultrasound in Med. and Biol.. 7 (1981) 2533265.

Acoutical damage to the rat brain 193

4. Chapman, I. Y., Macnally, N. A. & Tucker, S., Ultrasound-induced changes in rates of influx and efflux of potassium ion in rat thymocytes in vitro. Ultrasound Med. and Biol., 6 (1980) 47-58.

5. Taylor, K. J. W. & Pond, J., The effects of ultrasound of varying frequencies on rat liver. J. Pathol., 100 (1970) 287-293.

6. Taylor, X. J. W. & Pond, J. B., A study of the production of haemorrhagic injury and paraplagis in rat spinal cord by pulsed ultrasound of low megahertz frequencies in the context of the safety for clinical usage. Br. J. Radiol., 45 (1972) 343-353.

7. Prichard, J. W. & Shulman, R. G., Ann. Rev. Neurosci., 9 (1986) 61. 8. Fenstermacher, M. J. & Narayana, P. A., Znvest. Radiol., 25 (1990) 1034.