12 Fluoride Vol. 35 No. 1 12-21 2002 Research Report

HISTOLOGICAL CHANGES IN THE BRAIN OFYOUNG FLUORIDE.INTOXICATED RATS

YM Shivaraiashankara,' AR Shivashankara," P Gopalakrishna Bhat,bs Muddanna Rao.'s Hanumanth Raoo

Karnataka, India

SUMMARY: Wistar albino rats were exposed to 30 or 100 ppm fluoride (asNaF) in drinking water during their fetal, weanling, and post-weaning stagesuntil the age of ten weeks. Rats exposed to 30 ppm fluoride did not show anynotable alterations in brain histology, whereas rats exposed to 100 ppm fluo-ride showed significant neurodegenerative changes in lhe hippocampus,amygdala, motor cortex, and cerebellum. Changes included decrease in sizeand number of neurons in all the regions, decrease in the number of Purkinjecells in the cerebellum, and signs of chromatolysis and gliosis in the motorcortex. These histological changes suggest a toxic effect of high-fluoride in-take during the early developing stages of life on the growth, differentiation,and subcellular organization of brain cells in rats,Keywords: Albino rats, Amygdala, Brain histology, Cerebellum, Fluoride intoxication,

Hippocampus, Molor cortex, Neurodegenerative changes, Sodium fl uoride.

NTRODUCTION

Long-term intake of high levels of fluoride in humans causes neurologicalcomplications such as paralysis of limbs, vertigo, spasticity in extremities,and impaired mental acuity.r Fluoride is also known to cross the blood-brainbanier.2 Fluoride accumulation was observed in the brain of experimentalanimals exposed to chronic high-fluoride intake, and this accumulation in-creased as the drinking water fluoride levels increased.l Chronic fluoridetoxicity is also known to cause altered neuronal and cerebrovascular integ-rity,a abnormal behaviour pattems,3 and metabolic lesions in the brain,5-7 inexperimental animals.

In the present study, effects of chronic fluoride intoxication on brain cellstructure were assessed by exposing rats to 30 or 100 ppm fluoride indrinking water at the early stages of life and then studying the histology ofvarious brain regions.

MATERIALS AND METHODSAdult (4-6 month-old) wistar albino rats were used in the study. The rats

were fed with a standard pelleted diet (Hindustan Lever Ltd., India), andwere given water ad libitum. The animals were maintained under propertemperature, ventilation, and hygienic conditions.

Pregnant rats were divided into three groups: control, 30F, and 100F.Control rats received drinking water with 0.5 ppm fluoride, while the 30F

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and l00F ras rcceivcd 3Otively, in their &inting rrrtrougtou 6e bctaln ptrals on wcming ad rtret0.5 ppm,30 p, ud l(u eels. Tb o-rrcelold n= 9) goqe wre sificadranscadlUty sith 0.93 e\r er€ r€dwed-

The haic were fircd irof the hipocaqus, rylThe soctins w€s€ Focesssratrcd sectios were ots

ENo hisological cnaqps

T hen coryar€d to the ccnl00F groq, however, fur arios brain regions; the Ibellum showed neurodegrn

ln the hippocampus, msrmll nrrclans. and tfure wrrtratiom were seeo in crr&ntate gmrs sb regirrc o-d CAr sb regim. fich.nges armg the varic!hg-t€ro imate of high hrl9o.td ffrside acqm&6c hipoc*mfus. Ite pdlfoding of Varner et d vlC-{r sw of the hippocnpced to l#r fluaftbbr

\ieurodcgcoemtive chmgbclhrn were similar to d$Daor catcx, oanms in fr6ere sere also siEn< of ctot6erved chumtin cluqihi<n prcsence of ghc{irrs expoced to chrook irrqt- the hrtinjc Elb ir Ihfurl atd there sr a Lcrease in thc nurnbcr ofh"For Correspondence: YM Shivarajashankara, Dept. of Biochemistry, MR Medical Col-

lege, Gulba;ga-585105, Karnataka, India; E-mail: siivralsympyahoo.com. bDept. of Bio-chemistry, Kasturba Medical College, Manipal-5761'19, Karnataka, India. cDept. of Ana!omy, Kasturba Medical College, Manipal-576119, Kamataka, India. "Dept. of Biochemis-try, KBN Institute of Medical Sciences, Gulbarga-s8s104, Kamataka, India.

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Histological changes in the brain in fluoride intoxication 13

and 100F rats received 30 ppm and 100 ppm fluoride (from NaF), respec-tively, in their drinking water during the last (3'd) week of pregnancy andthoughout the lactation period. The litters were separated fiom the motherrats on weaning and were then exposed to the respective levels of fluoride(0.5 ppm, 30 ppm, and 100 ppm) in drinking water up to the age of 10ri,eeks. The ten-week-old rats ofcontrol (n: 15), 30F (n: 13), and 100F (n= 9) groups were sacrificed after light ether anaesthesia. They were perfusedtranscardially wirh 0.9Y:o saline followed by 10% formalin, and the brainslr ere removed.

The brains were fixed in 10% formalin, and 5-pm thick coronal sectionsof the hippocampus, amygdala, motor cortex, and cerebellum were taken.The sections were processed, and stained in 0.1% cresyl violet stain. Thestained sections were observed under a binocular light microscope.

RESULTS AND DISCUSSIONNo histological changes were seen in the brain regions of the 30F group

u hen compared to the corresponding brain regions ofthe control group. Thel00F group, however, showed significant changes in the histology of ther arious brain regions; the hippocampus, amygdala, motor cortex, and cere-lellum showed neurodegenerative changes (Figures l-6).

In the hippocampus, neurons were shrunken and darkly stained with a.mall nucleus, and there was a decrease in the cell number. Histological al-:erations were seen in cornu ammonis-l (CAr), comu ammonis-4 (CAa), andJentate gyrus sub regions of the hippocampus (Figures 1-3), but not in CAz.rnd CA3 sub regions. The hippocampus showed the most pronounced:hanges among the various brain regions of 100F rats. In rats exposed toong-ierm intake of high levels of fluoride in drinking water, Mullenix er a/r:eported fluoride accumulation in important regions of the brain, especially:re hippocampus. The pattem ofchanges seen in this study are similar to the:rndings of Varner et ala who reported cellular abnormalities in the CAr and. .\1 areas of the hippocampus, and not in CAz and CA:, in adult rats ex-:,rsed to 1-ppm fluoride in drinking water for 52 weeks.

\eurodegenerative changes seen in the amygdala, motor cortex, and cere--cllum were similar to those seen in the hippocampus (Figures 4-6). In the':otor cortex, neurons in the superficial layers were the most affected, and:rere were also signs of chromatolysis and gliosis (Figure 5). Yamer et ala'bserved chromatin clumping, enhanced protein staining, pyknosis, vacuo-.uion, presence of ghosrlike cells, and decreased neuronal density in adult:rs exposed to chronic intake of l-ppm fluoride in drinking water. In our.i ork, the Purkinje cells in the cerebellum were the most affected cell popu-:rion. and there was an increase in the number of granular cells but a de--rease in the number of Purkinje cells and molecular cells (Figure 6).

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Earlier, Zhavoronkov8 reported foci of demyelination in the cortex andsubcortical areas, a decrease in the number of Purkinje cells in the cerebel-lum, swelling and irregular staining of the Nissl substance, and pyknosis oneurons in experimental animals subjected to fluoride intoxication.

In the present studn mother rats were exposed to high levels offluoride ithe last week of pregnancy and during lactation, This was d. one to ensurethat the mother's body system became laden with fluoride so the litterswould receive fluoride through the mother. Later, the litters were weanedand exposed directly to fluoride in drinking water. Thus, the yormg rats weresubjected to chronic fluoride intoxication during the developing stageslife. Li et aP reported a lower intelligence quoti€nt (.Q.) in childrenin fluoride-endemic areas and suggested that the effect of exposure to highlevel of fluoride on intelligence probably occurs at an early stage of devel-opment of the embryo and infant when differentiation of brain cells is occur-ring and development is most rapid. Eartier, Mullenix et aF observed ab-normal behaviour pattems in rats exposed to intake ofhigh levels ofduring prenatal, weanling, or adult stages. Itrowever, we did not ichanges in the rat behaviour pattems that might have occurred in our study.

In conclusion, we find that chronic fluoride intoxication in the earlyof life caused marked neurodegenerative changes in the brain of rats.changes may form the neural basis for impaired leaming and memory, ab-normal behaviour pattems, and disturb€d overall body physiology.

ACKNOWLEDGEMENT

We are grateful to Dr B Mallikarju4 Dean, M R Medical College, Gul-barg4 for his zupport and encouragement ofthis research project.

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lemma. Lawrence. Kansas: Coronado Pness:1978.2 Geeraerts F, Gijs G, Finne E,'Grokaert R Kinetics of fluoride penetration

liver and brain. Fluoride | 986:19:108-12.3 Mullenix PJ, Denbesten PK SchEior A" Kernan WJ. Neurotoxicity of

fluoride in rats. Neurotoxicol T€ratol.1995:,17l.169-77.4 Vamer JA, Jensen KF, Horwath Vr', Issacson RL. Chronic administration

aluminium or sodium fluoride to rats in ddnking water: alterations in neuroand cerebrovascular integrity, Brain Res 1998;784:284-98.

5 Shashi A. Studies on alterations in brain lipid metabolism followingmental fluorosis. F\toide 1992;25:77 -84.

6 Shashi A, Singh JP, Thapar SP. Effect of long-term administration ofon levels of proteins, free amino acids and RNA in rabbit bnain. Fluoride1994;27 :155-9 .

7 Vani ML, Reddy KP. Effect of fluoride accumulation 04 some enzymesbrain and gastrocnemius mlscle of mice. Fluoride 2000;33 : 1 7-26.

Fluoide 35 (1) 2002

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cortex andhe cerebel-x knosis of

I luonde ln

I tO ensurethe litters

re weanedB rats were; stages ofiren livinglre to highl of devel-ls rs occur-sen ed ab-of fluorideinvestigatexlr study.all-v- Srages

r:at s. These:nory, ab-

llege, Gul-

r great di-

n!'tration in

I rrf sodium

istration ofin neuronal

ing experi-

of fluoriden. Fluoride

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Histological changes in the brain in fluoride intoxication 21

Zhavoronkov AA. Non-skeletal forms of fluorosis. Arch Pathol 1977:.39.83-9 1 .Li XS, Zhi JL, Cao RO. Effect of fluoride exposure on intellisence in children.Fluoride 1995;28: I 89-92.

Published by the International Society for Fluoride Research::::lral Offlce: 727 Brighton Road, Ocean View, Dunedin 9051, New Zealand

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