Iron deficiency is the most common nutritional deficiency worldwide, affecting approximately two billion individuals . Its prevalence is the highest in population segments at peak rates of growth, such as infants, young children, and pregnant women .
The WHO considers iron deficiency anemia (IDA) to be the number one nutritional disorder in the world. It remains a considerable public health problem, with major consequences for human health as well as social and economic development . The low intake of total dietary iron and poor iron absorption because of iron-absorption-inhibiting factors, such as tannins in tea, were identified as the common causes of IDA .
In the last few years, in Egypt, considerable attention has been paid to the hazardous effects of IDA on health, particularly on the health of children. Hence, many public health programs are being implemented to reduce the prevalence of (IDA) such as fortification of wheat flour and supplementation of food with an iron element. However, these programs remain controversial .
Iron deficiency may produce an impairment in iron-dependent enzyme systems, affecting the metabolism and the kinetics of the rapidly dividing oral epithelial cells. Iron deficiency may also reduce the cell-mediated immune response, phagocytosis and induce inadequate antibody production .
The oral cavity is often one of the first sites where nutritional deficiencies including iron deficiency can be clinically noted . The clinical manifestations of iron deficiency include glossal pain, dysphagia, and a smooth red tongue .
Glossodynia (burning mouth syndrome) is also associated with iron deficiency, where the tongue is sensitive to hot and spicy food .
Generally, the oral mucous membrane is highly sensitive and its integrity is maintained by complex interacting factors that require an adequate supply of nutrients including iron .
Aim of the work
There are few histological studies on the changes on the tongue in IDA, although many clinical findings, in the oral cavity and the tongue, have frequently been reported. Thus, the present work aimed to study the possible histological changes that may occur in the tongue of young rats, which were fed an iron-deficient diet for 6 weeks. It also aimed to study the role of a balanced diet, containing the necessary daily requirement of iron, either alone or with daily oral therapeutic iron supplementation to ameliorate these changes.
Materials and methods
Two forms of diets were prepared at the Nutritional Department, National Research Center, Giza, Egypt: a balanced diet that included the daily iron requirements and an iron-deficient diet. Both types of diets were formulated to provide all the recommended nutrients as follows (g/100g): protein: 10, fat: 10, carbohydrates: 74.4, fibers: 1, salt mixture: 3.5, vitamin mixture, water-soluble vitamin: 1; and methionine: 0.1 . The salt mixture in the balanced diet contained 36mg iron/kg diet, whereas the iron-deficient diet contained 5mg iron/kg diet . Iron used was in the form of ferrous sulfate. Fat-soluble vitamins (A, D, E, and K) were provided orally to all animals in the study at a dose of 0.1ml/rat/week using an intragastric tube.
For oral iron supplementation, ferrous sulfate tablets (105mg elemental iron) were purchased from Teofarma, Italy. The tablets were crushed and dissolved in distilled water. Iron supplementation was provided to the animals orally by an intragastric tube at a dose of 9.45mg iron/kg body weight once daily .
Thirty young male albino rats (4 weeks old) were used in the present study. Their weights ranged from 45 to 50g. They were housed in wire mesh cages, three animals per cage. The rats were allowed to consume water and diet ad libitum. They were divided into two main groups:
Group I: The control group
It included 12 rats that were fed a balanced diet. It was further subdivided into two subgroups of six animals each.
Subgroup IA included the rats that were allowed to live for 6 weeks from the beginning of the experiment.
Subgroup IB included the rats that were sacrificed 8 weeks from the beginning of the experiment.
Group II: The experimental group
It included 18 rats that were fed an iron-deficient diet. It was further subdivided into three subgroups of six animals each:
Subgroup IIA included the rats that were fed an iron-deficient diet for 6 weeks and were then sacrificed.
Subgroup IIB included rats that were fed an iron-deficient diet for 6 weeks, followed by a balanced diet for 2 weeks.
Subgroup IIC included the rats that were fed an iron-deficient diet for 6 weeks. Thereafter, the animals were fed a balanced diet and were also provided oral iron supplementation at a dose of 9.45mg iron/kg body weight once daily by an intragastric tube for 2 weeks.
At the end of the experiment, the animals were weighed before being sacrificed. Just after killing, blood samples were collected from each animal by heart puncture. The levels of hemoglobin (Hb) and serum iron were determined for each blood sample.
Note: In subgroups IIB and IIC, blood samples were also taken at the end of the sixth week from the beginning of the experiment. They were taken from the tail vein to confirm the occurrence of IDA before the treatment.
From each animal, the tongue was removed and the anterior two-thirds of the tongue was dissected. Then, each specimen was cut longitudinally into two halves. One half was fixed in 10% formol saline and processed for the preparation of serial paraffin sections (5-μm-thick). The sections were stained by H&E stain.
The other halves of the specimens were cut into small pieces, washed by phosphate-buffered saline, and fixed in a phosphate-buffered gluteraldehyde solution. The specimens were processed for examination using a Philips Scanning Electron Microscope (XL30; Philips, Amsterdam, the Netherlands) at the Scanning EM Unit of the Anatomy Department, Faculty of Medicine, Ain Shams University.
For the morphometric study, the Image Analyzer (Leica Q 500 MC program) in the Histology Department, Faculty of Medicine, Ain Shams University was used to determine the mitotic index of the epithelial covering of the tongue by counting the dividing cells (any stage of mitosis) in the basal and parabasal layers/100 cells. Five serial H&E-stained sections were used in each animal in each subgroup.
For the statistical study, the mean body weight of the animals, the Hb level, serum iron level, the mitotic index, and the mast cell number were calculated in the different subgroups. The Student t-test was used to compare the data and the P value was calculated using the SPSS program, version 17. A P value < 0.05 was considered significant.
Subgroup IA: rats that were fed a balanced diet for 6 weeks: Histological examination of H&E-stained sections showed that the dorsal and ventral surfaces of the tongue were covered by a mucous membrane. This was composed of keratinized stratified squamous epithelium and a corium of connective tissue. The dorsal surface of the anterior two-thirds of the tongue was characterized by the presence of tongue papillae that showed a thick epithelial covering with a thick keratin layer. The slender filiform papillae were the most numerous, whereas the fungiform papillae were few and interposed among the filiform ones. On the upper surface of the fungiform papillae, taste buds could be seen (Fig. 1, inset).
The mucous membrane of the ventral surface lacked lingual papillae (Fig. 2). Different phases of mitosis could be detected in the cells in the basal and parabasal layers of the epithelium (Fig. 3).
Underneath the epithelial covering of the dorsal and ventral surfaces of the tongue, there was a connective tissue layer continuous with the perimysium and endomysium of the muscles (Figs 1 and 2).
The skeletal muscle fibers constituted the main bulk of the tongue. They were crowded and composed of interlacing bundles that were running in longitudinal, transverse, and vertical directions (Fig. 4).
On scanning electron microscopical examination, the dorsal surface of the tongue was covered by numerous filiform papillae. They revealed an elongated conical shape with intact tapering tips. They were slightly curved, with their tips pointed toward one direction. Fungiform papillae were observed sporadically between the filiform ones. They were short and broad in shape and had a flattened, smooth hemispherical upper portion. Taste pores could be seen on the upper surface of the fungiform papilla (Figs 5 and 6).
Subgroup IB: rats that were fed a balanced diet for 8 weeks: Histological examination of different sections in this subgroup showed features that were very similar to those of subgroup IA.
Group II: The experimental group
Subgroup IIA: rats that were fed an iron-deficient diet for 6 weeks: H&E-stained sections showed variable but focal histological changes. The lingual papillae showed apparent shortening and broadening when compared with the control group (Fig. 7). Focal areas of flattening of the dorsal surface of the tongue were also observed (Fig. 7).
Nuclear changes could be observed in the basal and parabasal layers of the epithelial covering the dorsal surface of the tongue. For instance, condensation and margination of the chromatin materials against the nuclear membrane were detected, resulting in a crescent-like appearance (Fig. 8). Moreover, pyknotic nuclei could also be observed (Fig. 8).
The epithelial lining of the ventral surface also showed an apparent reduction in thickness (Fig. 9). The keratin layer of both the dorsal and the ventral surfaces of the tongue appeared thin, discontinuous, and detached in some areas, with loss of integrity (Figs 7 and 9).
Congested blood vessels as well as mononuclear cells appeared to infiltrate the connective tissue underneath the epithelium (Fig. 9). The skeletal muscle fibers were apparently shrunken and widely separated. A few rounded empty spaces were also observed in between the muscle fibers (Fig. 10). Clear empty spaces could be observed near the congested blood vessels of the lamina propria (Fig. 9).
Scanning electron microscopical examination revealed a noticeable atrophy of the tongue papillae, from being short to being completely absent in focal areas (Fig. 11). They were widely separated and irregularly arranged in different directions (Fig. 12). The upper portions of some papillae appeared bisected (Figs 13 and 14). Furthermore, some filiform papillae showed desquamation of their epithelial lining and small rounded structures could be observed on these exposed areas (Fig. 15).
Some fungiform papillae had a rough hemispherical upper portion with indistinct taste pores (Fig. 13), whereas others showed complete loss of their upper portions (Fig. 14).
Subgroup IIB: rats that were fed an iron-deficient diet for 6 weeks, followed by a balanced diet for 2 weeks: Examination of H&E-stained sections showed that the tongue papillae were apparently shorter than those in the control group. The keratin layer appeared separated and discontinuous in focal areas. Although the epithelial lining of the ventral surface appeared thicker than that in subgroup IIA, it was still apparently thinner than that in the control group (Fig. 16, inset). The skeletal muscle fibers showed small rounded empty spaces in between muscle fibers. Their bundles appeared widely separated with congested blood vessels between them (Fig. 17).
Scanning electron microscopical examination showed that in some areas, the filiform papillae were deformed. Some papillae showed blunted tips whereas others still had a bisected upper portion (Fig. 18).
Subgroup IIC: rats that were fed an iron-deficient diet for 6 weeks, followed by a balanced diet and iron supplementation for 2 weeks: Examination of H&E-stained sections showed that the tongue surfaces (dorsal and ventral) were very similar to those in the control group (Fig. 19, inset). The muscle fibers appeared arranged in different directions with narrow spaces between them (Fig. 20).
Scanning electron microscopy examination showed that the tongue papillae were regularly arranged. The filiform papillae appeared long and conical in shape with intact tips. Fungiform papillae were interposed in between them (Fig. 21).
The biochemical results
There was a highly significant decrease in both the Hb and the serum iron levels after 6 weeks of administration of an iron-deficient diet in subgroups IIA, IIB, and IIC when compared with their corresponding control group (subgroup IA) (Table 1). Meanwhile, in comparison with the control subgroup IB, these parameters showed a significant decrease in subgroup IIB and a nonsignificant change in subgroup IIC (Table 2).
The body weight
There was a highly significant decrease in the mean body weight of animals in subgroup IIA as compared with the control subgroup IA. In subgroups IIB and IIC, the mean body weight of the animals still showed a highly significant decrease and a significant decrease, respectively, as compared with the control group (subgroup IB) (Table 3).
The morphometric results
In terms of the mitotic index, a highly significant decrease was observed in subgroup IIA when compared with control subgroup IA. Meanwhile, the mitotic index in subgroups IIB and IIC showed a significant decrease and a nonsignificant change, respectively, when compared with the control subgroup IB (Table 4).
In the present study, young rats that were fed an iron-deficient diet for 6 weeks (subgroup IIA) showed a highly significant reduction in their Hb and serum iron levels when compared with the control group. These biochemical parameters indicated the occurrence of IDA. The results were in agreement with those obtained by many authors who observed that the reduction in the Hb level in rats fed an iron-deficient diet had started from the second week onward [12,14].
In the current study, there was a highly significant decrease in the body weight of animals in subgroup IIA in comparison with the control group.
This agreed with the results of many authors [12,14] who demonstrated the negative impact of iron deficiency on growth and development. They also found that the daily mean food intake was statistically significantly lower in rats fed an iron-deficient diet when compared with the control rats. They attributed the poor growth rates to anorexia and malnutrition.
In the present study, structural changes in the tongues of rats in subgroup IIA were also observed. An apparent shortening and broadening of the lingual papillae were observed. The epithelial covering of both the dorsal and the ventral surfaces showed an apparent reduction in thickness. The keratin layer showed loss of integrity and appeared thin and discontinuous.
Moreover, the morphometric and statistical results revealed a highly significant decrease in the mitotic index of the cells of the epithelial covering of the tongue as compared with the control group. Thus, this decrease in the thickness of the epithelial covering of the tongue might be a result of the higher rate of cell exfoliation compared with the rate of mitosis.
This was in agreement with the result obtained by Umbreit , who reported that iron is indispensable for DNA synthesis and consequently cell division. Hence, iron deficiency could interfere with the normal mitotic divisions of the cells.
It was evident that in the absence of iron, cellular respiration was affected and the cell was metabolically compromised. Consequently, the energy that was derived from cellular respiration and oxidative phosphorylation was markedly affected and the cells could not serve their functions or divide .
Moreover, iron deficiency resulted in defects in DNA and RNA synthesis as enzymes were drastically affected, such as ribonucleotide reductase, which is responsible for converting ribonucleotide into deoxyribonucleotide (RNA to DNA). This enzyme requires iron for its optimum function. Thus, in absence of iron, this reaction cannot proceed and DNA is not produced .
In the current study, focal areas of flattening of the dorsal surface of the tongue were found. Some papillae appeared with bisected tips and, in other papillae, areas of epithelial desquamation were also observed.
Similar results were reported in a preliminary study by Scott et al.  on human tongue biopsies from individuals with IDA. They attributed the atrophy of the lingual papillae to the decrease in the number and size of the cells together with the degenerative changes in the epithelial lining of the tongue.
Furthermore, atrophy of the lingual papillae was assumed by many authors to be the reason for the smooth appearance of the tongue on clinical examination in patients with IDA [4,17].
In the current study, the nuclei of some cells in the basal and parabasal layers of the epithelium showed condensation and margination of their nuclear chromatin materials against the nuclear membrane, resulting in a crescent-like appearance. This could be one of the nuclear apoptotic changes described previously by Bursch et al. . They also suggested that iron deficiency might enhance apoptotic changes in the cells of tongue epithelium.
It was reported that iron deprivation resulted in apoptotic induction through mitochondrial alterations .
The presence of mononuclear cellular infiltration in the lamina propria of the tongue together with congested blood vessels was observed in the current study. These results, in addition to thinning of the epithelial covering of the tongue, were considered to be inflammatory changes affecting the tongue (glossitis) . It is known that glossitis is one of the clinical manifestations of patients with IDA [4,10,17].
The mechanisms by which the iron deficiency could induce inflammation and tissue damage have been discussed extensively. Some investigators have reported an elevation in lipid peroxidation in IDA that could be because of the increased fragility of mitochondrial membranes [21,22]. In addition, it was evident that gastrointestinal upregulation of iron absorption during iron deficiency increases copper absorption as well. Copper can participate in the generation of reactive hydroxyl radicals that can result in the damage of lipid and DNA .
Furthermore, in IDA, there is a reduction in serum retinol and accumulation of vitamin A in the liver as retinyl esters. This reduction could be because of the impaired activity of the converting enzyme (hepatic retinyl ester hydrolase), which is an iron-dependent enzyme .
Vitamin A helps the mucous membrane to remain healthy by controlling the growth and differentiation of cells; thus, vitamin A deficiency impairs the oral and tongue mucosa structures .
In the current study, areas of epithelial desquamation in some lingual papillae with small rounded structures present on these exposed areas could be observed on scanning electron microscopic examination. These structures might be bacterial colonies .
The increased susceptibility to oral infections and recurrent mouth ulcers in cases of IDA has been documented, whereas several enzymes are involved in bactericidal action and those involved in the production and breakdown of hydrogen peroxide are iron-containing enzymes. Thus, iron deficiency could affect their functions and consequently repeated infections might occur [3,4].
Glossal pain and glossodynia (burning tongue) are common complaints in patients with IDA [9,10,17]. This could be because of atrophy of tongue epithelium, resulting in the disturbance of the underlying nerves, thus leading to a painful tongue .
In the current study, filiform and fungiform papillae could be observed in the anterior two-thirds of the dorsal surface of the control rat tongue. Fungiform papillae containing taste buds in their apical poles were detected. The circumvallate papillae were not observed in the present study because there is only one circumvallate papilla in rat tongue that is placed posteriorly in the midline .
Deformity of the fugiform papillae with the destruction of the upper surface of some of them was observed in the tongues of iron-deficient rats. This could be responsible for the taste disturbance in an iron deficiency state .
In the present study, skeletal muscles of the tongue appeared shrunken and widely separated in rats that were fed an iron-deficient diet. Similar changes were also described by Jarvinen et al.  in cases of atrophic glossitis. The arrangement of the muscles gives the tongue considerable mobility to manipulate food around the mouth for efficient fragmentation and for moving fragmented food backward before swallowing . Affection of the tongue muscles might explain the difficulty in swallowing in patients with IDA . Moreover, in rats with IDA, there were altered skeletal muscle functions .
Moreover, there were small rounded empty spaces in between the muscle fibers, most probably fat cells. The results of a previous study suggested that there was a more generalized impairment in mitochondrial oxidative capacity in iron deficiency . This could impair the ability of muscle to oxidize fat, resulting in lipid accumulation .
The results of the current study showed that rats with IDA that received a balanced diet alone for 2 weeks (subgroup IIB) showed a mild improvement in the histological changes of the tongue. The levels of Hb and serum iron still showed a significant decrease as compared with the control subgroup IB.
However, daily oral iron supplementation together with a balanced diet (subgroup IIC) resulted in a marked improvement in the structure of the tongue, which appeared very similar to the control group.
The biochemical parameters showed a nonsignificant difference as compared with the corresponding control group (subgroup IB). These results were in agreement with those of Lynch et al. , who reported that iron supplementation was indicated in the treatment of IDA. They added that diet alone cannot restore deficient iron levels to normal within an acceptable time.
Furthermore, the goals of providing oral iron supplements are to supply sufficient iron to restore normal storage levels of iron and replenish Hb deficits .
It was evident that ferrous iron salts are the best-absorbed forms of iron supplementation .
It was recommended that physicians monitor the effectiveness of iron supplements through assessments of Hb and ferritin levels. The authors also added that the Hb level increases within 2–3 weeks of starting iron supplementation .
It was also reported that daily supplementation was more effective than weekly supplementation to increase Hb and ferritin levels, especially in school-age children .
Conclusion and recommendation
It was concluded that an iron-deficient diet induced structural changes in rat tongue. These changes might be responsible for the glossal pain and smooth red tongue that were described in clinical studies in cases of IDA.
These changes may also compromise dietary intake and worsen the nutritional condition of the patient. The daily oral iron supplementation, together with the intake of a balanced diet, resulted in restoration of the tongue structure faster than the intake of a balanced diet alone.
As deficient iron intake is a contributing factor in the development of IDA, iron-rich food such as cabbage, spinach, potato, tomato, eggs, chicken, and beef should be consumed. Moreover, oral iron supplementation remains an important strategy for the prevention of IDA in individuals at an increased risk such as infants, young children, and pregnant women.
Table. No title avai...Image Tools
Conflicts of interest
There is no conflict of interest to declare.
Walter PB, Knutson MD, Paler Martinez A, Lee S, Xu Y, Viteri FE, Ames BN. Iron deficiency and iron excess damage mitochondria and mitochondrial DNA in rats. Proc Natl Acad Sci USA. 2002;99:2264–2269
Agarwal T, Kochar GK, Goel S. Impact of iron supplementation on anemia during pregnancy. Ethnomed. 2008;2:149–151
Umbreit J. Iron deficiency: a concise review. Am J Hematol. 2005;78:225–231
Ghosh K. Non haematological effects of iron deficiency – a perspective. Indian J Med Sci. 2006;60:30–37
Winham DM, Harrison GG, Galal OM, El Tobgui M. Anemia and infection in school-aged Egyptian children. Ecol Food Nutr. 2004;43:29–40
Van Wyk C, Steenkamp V. Host factors affecting oral candidiasis. S Afr J Epidemiol Infect. 2011;26:18–21
Navia JM. A new perspective for nutrition: the health connection. Am J Clin Nutr. 1995;61:407S–409S
Osaki T, Ueta E, Arisawa K, Kitamura Y, Matsugi N. The pathophysiology of glossal pain in patients with iron deficiency and anemia. Am J Med Sci. 1999;318:324–329
Pierro VS, Maia LC, Primo LG, Soares FD. Case report: the importance of oral manifestations in diagnosing iron deficiency in childhood. Eur J Paediatr Dent. 2004;5:115–118
Lu SY, Wu HC. Initial diagnosis of anemia from sore mouth and improved classification of anemias by MCV and RDW in 30 patients. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2004;98:679–685
Pugh TD, Klopp RG, Weindruch R. Controlling caloric consumption: protocols for rodents and rhesus monkeys. Neurobiol Aging. 1999;20:157–165
Fernandes MI, Galvào LC, Bortolozzi MF, Oliveira WP, Zucoloto S, Bianchi ML. Disaccharidase levels in normal epithelium of the small intestine of rats with iron-deficiency anemia. Braz J Med Biol Res. 1997;30:849–854
Paget G, Barnes J Evaluation of drug activity. 1964 London Academic Press
Wayhs MLC, Patrício FSR, Amancio OMS, Pedroso MZ, Fagundes Neto U, Morais MB. Morphological and functional alterations of the intestine of rats with iron-deficiency anemia. Braz J Med Biol Res. 2004;37:1631–1635
Beard JL. Iron biology in immune function, muscle metabolism and neuronal functioning. J Nutr. 2001;131(Suppl 2):568S–580S
Scott J, Valentine JA, St Hill CA, West CR. Morphometric analysis of atrophic changes in human lingual epithelium in iron deficiency anaemia. J Clin Pathol. 1985;38:1025–1029
Touger Decker R. Oral manifestations of nutrient deficiencies. Mt Sinai J Med. 1998;65:355–361
Bursch W, Ellinger A, Kienzl H, Török L, Pandey S, Sikorska M, et al. Active cell death induced by the anti-estrogens tamoxifen and ICI 164 384 in human mammary carcinoma cells (MCF-7) in culture: the role of autophagy. Carcinogenesis. 1996;17:1595–1607
Koc M, Nad'ová Z, Truksa J, Ehrlichová M, Kovár J. Iron deprivation induces apoptosis via mitochondrial changes related to Bax translocation. Apoptosis. 2005;10:381–393
Jarvinen J, Arja K, Pesonen E. Histoquantitative study of inflamed tongue mucosa. Eur J Oral Sci. 1991;99:424–430
Uehara M, Chiba H, Mogi H, Suzuki K, Goto S. Induction of increased phosphatidylcholine hydroperoxide by an iron-deficient diet in rats. J Nutr Biochem. 1997;8:385–391
Knutson MD, Walter PB, Ames BN, Viteri FE. Both iron deficiency and daily iron supplements increase lipid peroxidation in rats. J Nutr. 2000;130:621–628
Allen LH. Iron supplements: scientific issues concerning efficacy and implications for research and programs. J Nutr. 2002;132(Suppl):813S–819S
Biesalski HK, Nohr D. New aspects in vitamin a metabolism: the role of retinyl esters as systemic and local sources for retinol in mucous epithelia. J Nutr. 2004;134(Suppl):3453S–3457S
Motoyama AA, Watanabe I, Semprini M, Lopes RA, Iyomasa MM, Mizusaki CI. Scanning electron microscopy of the rat tongue mucosa with special attention to the bacteria on epithelial cell membranes. Braz Dent J. 1999;10:11–14
Iwasaki SI, Yoshizawa H, Kawahara I. Study by scanning electron microscopy of the morphogenesis of three types of lingual papilla in the rat. Anat Rec. 1997;247:528–541
Stevens A, Lowe J Human histology. 19972nd ed. Philadelphia Mosby 2nd ed.
Thompson CH, Green YS, Ledingham JG, Radda GK, Rajagopalan B. The effect of iron deficiency on skeletal muscle metabolism of the rat. Acta Physiol Scand. 1993;147:85–90
Hagler L, Askew EW, Neville JR, Mellick PW, Coppes RIJ, Lowder JFJ. Influence of dietary iron deficiency on hemoglobin, myoglobin, their respective reductases and skeletal muscle mitochondrial respiration. Am J Clin Nutr. 1981;34:2169–2177
Morino K, Petersen KF, Shulman GI. Molecular mechanisms of insulin resistance in humans and their potential links with mitochondrial dysfunction. Diabetes. 2006;55(Suppl 2):S9–S15
Lynch S, Stoltzfus R, Rawat R. Critical review of strategies to prevent and control iron deficiency in children. Food Nutr Bull. 2007;28(Suppl):S610–S620
Kumpf VJ. Invited review: parenteral iron supplementation. Nutr Clin Pract. 1996;11:139–146