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.
Conflicts of interest
There is no conflict of interest to declare.
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Keywords:© 2012 The Egyptian Journal of Histology
iron deficiency; tongue and iron supplementation