Histological and ultrastructural aspects of the tongue in undernourished rats

Summary There are few published studies on the effects of protein-caloric undernourishment on the oral mucosa. Objectives The objective of this study is to verify the histological and ultrastructural aspects of the tongue mucosa in protein-caloric undernourished adult rats. Material and Methods A clinical experimental study was done in thirty Wistar rats, 15 controls and 15 with protein-caloric undernourishment. The last group received ration in small amounts, with a reduced casein content, during 45 days. Rats were weighed every 3 days, from the first (90 days of life) to the last day of a 45-day dietary period, when they were sacrificed. Plasma was used for protein electrophoresis and their tongues were prepared for light and scanning electron microscopy. Analysus of variance and Student's t-test were used for statistical analysis. Results A significant decrease in weight and in plasma proteins was found in protein-caloric undernourished rats compared to the control group. Histological findings revealed no differences between the two groups and there were no statistically significant differences in the filiform papilla count under the scanning electron microscopy. Conclusion Protein-caloric undernourishment does not cause alterations in the tongue mucosa of adult rats.

There are few published studies on the effects of proteincaloric undernourishment on the oral mucosa. Objectives: The objective of this study is to verify the histological and ultrastructural aspects of the tongue mucosa in protein-caloric undernourished adult rats. Material and Methods: A clinical experimental study was done in thirty Wistar rats, 15 controls and 15 with protein-caloric undernourishment. The last group received ration in small amounts, with a reduced casein content, during 45 days. Rats were weighed every 3 days, from the first (90 days of life) to the last day of a 45-day dietary period, when they were sacrificed. Plasma was used for protein electrophoresis and their tongues were prepared for light and scanning electron microscopy. Analysus of variance and Student's t-test were used for statistical analysis. Results: A significant decrease in weight and in plasma proteins was found in protein-caloric undernourished rats compared to the control group. Histological findings revealed no differences between the two groups and there were no statistically significant differences in the filiform papilla count under the scanning electron microscopy. Conclusion: Protein-caloric undernourishment does not cause alterations in the tongue mucosa of adult rats.

INTRODUCTION
Although clinical findings in the mouth such as tongue mucosa atrophy, loss of papillae, and angular cheilitis, are frequently related to nutrition, there are few studies on the effect of protein-energy malnutrition over the oral mucosa and the tongue 1-4 .
Winkler and Nakamoto in 1982 5 studied the effects of prenatal malnutrition on tissue development in the mouth of newborn rats. They described histological alterations on the tongue such as reduced cell numbers and the increased size of these cells. Malnutrition also affected incisor and molar dental bacteria. Results suggested that adequate protein intake was extremely important for the healthy development of teeth and the tongue. Aldred et al. in 1989 6 reported an increase in yeasts in the mouth of malnourished children, but found no other clear clinical signs.
The aim of this paper is to study the effect of protein-energy malnutrition on the mucosa of the tongue of adult rats, using optical and scanning electron microscopy.

MATERIAL AND METHODS
This is an experimental study made on thirty "WISTAR" rats aged 90 days and weighing between 180 and 220 grams. Animals were placed in individual cages measuring 40 x 20 x 13 cm each, containing sawdust and water with no restrictions.
Animals were randomly divided into two groups each containing 15 rats. The first group (control) received ration composed of casein (18%), corn (4%), salt (4%), multivitamins (1%), benzoate (0.1%), oil (8%), sugar (10%), corn starch excipient qs (100%). The second group (with protein-energy malnutrition) received a similar ration but with changes in the relative components of casein (1%), given as ration weighing 6 grams/day. Rats were weighed every 3 days from the first day (age 90 days) until the final day of the 45-day diet period. At this point, having been weighed, the animals were guillotined. Blood was collected and plasma was obtained by centrifugation for protein electrophoresis.
The tongue was sectioned and completely removed. The anterior region of the tongue was sectioned for light and electron microscopy.
Fragments were included in paraffin for microtome sectioning and hematoxylin-eosin (HE) stained. Slides were studied under a common optical microscopy at 50 and 400 times amplification. The following parameters were assessed: epithelial layer uniformity or lack thereof, the presence or absence of an inflammatory infiltrate in the lamina propria, and areas with or without filiform and fungiform papillae in the anterior region of the tongue. Uniformity was considered as the regular distribution of layers with similar cell maturity.
Seven cases in each group (control and malnourished) were assessed by electron microscopy. The number of filiform papillae was one of the study parameters; counts were obtained from electron microscopy pictures taken using VP 120 black-and-white film. Inclusion criteria were: the number of whole filiform papillae (which included a base and a conical projection) counted on pictures magnified 100 times, measuring 9 x 9 cm, using a 2.3 cm 2 area, in each case counting 5 to 10 regions. Tangential papillae in the left and lower borders were included; tangential papillae in the right and upper borders were excluded; regions with fungiform papillae were excluded 7 .
Statistical analysis included the analysis of variance to compare average rat weight, protein electrophoresis and the papillary count. Student's T-test was used to study the difference between two independent samples 8 with a 0.05 or 0.5% significance level.

RESULTS
Weight increased in the control group and decreased in the protein-energy malnutrition group after 45 days. Statistical analysis revealed a significant difference between both samples on the 45th day, as shown on Chart 1.
The plasma protein electrophoresis profile disclosed a significant reduction in total plasma proteins in the protein-energy malnutrition group on the 45th day compared to controls (Tables 1 and 2).
Common light microscopy histology revealed no differences between the control group and the proteinenergy malnutrition group for the parameters we studied (Figures 1 and 2).
Common light microscopy histology of the tongues of rats in both groups showed stratified epithelium with uniform layers in 15 cases. Neither group had an inflammatory infiltrate or areas of papillary loss in the tongue. Keratinization of the ventral surface of the tongue was seen in 2 cases in the control group (with thickening of the keratin layer in 1 control group case) and in no case of the protein-energy malnutrition group. Chart 1. Histogram of rat body weight averages in the control group and the protein-energy malnutrition during the 45-day period.   There were no statistically significant differences in the filiform papillae count by electron microscopy on the tongue in both the control group (7 cases) and the protein-energy malnutrition group (7 cases), as seen on Tables 3, 4 and 5 (Figures 3 and 4).

DISCUSSION
Malnutrition is a significant public health problem in the world; protein-energy malnutrition is one of the most severe forms of this condition. It affects mostly children in poorer coutries, where the prevalence is estimated as 1/3 of children between birth and five years of age 9 .
The World Health Organization defines proteinenergy malnutrition as "the range of pathological conditions arising from a deficiency of cell intake, transport or use of nutrients, commonly associated with infection, occurring with greater frequency in infants and pre-school children" 10 .
Protein-energy malnutrition causes a variety of clinical conditions, the most apparent being effects on body measurements and weight as a whole or for specific organs. Malnutrition develops in a sequence starting from exclusive protein deficiency (kwashiorkor) to protein-energy deficiency (marasmus) 11 . In man, nutritional deficiency is never an exclusively caloric or protein deficiency; both are usually associated.
Clinically there is a strong association between protein-energy malnutrition and hypovitaminosis, and   glossities and stomatitis [1][2][3][4] . In out study the 45-day proteinenergy deficient diet effectively produced malnutrition in rats aged 90 days, as shown in the statistically significant differences between the control group and the proteinenergy malnutrition group; there was both weight loss and a reduction in total proteins. However, the histological study of the tongue of protein-energy malnutrition rats, both under common optical microscopy and electron microscopy did not reveal changes such as glossitis or loss or filiform papillae. There are no published papers on the effects of protein-energy malnutrition specifically on the mucosa of the mouth or tongue. There is one paper describing changes in prenatal malnutrition 5 . In contrast, there are many reports on the clinical, histological and ultra-structural changes of the tongue mucosa in iron deficiency, particularly during pregnancy and the postnatal period [1][2][3][4]12 , and vitamin