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International Journal of Morphology

versión On-line ISSN 0717-9502

Int. J. Morphol. v.21 n.2 Temuco  2003

http://dx.doi.org/10.4067/S0717-95022003000200008 

Int. J. Morphol., 21(2):143-148, 2003.

PROGRESSIVE EFFECTS OF DIABETES MELLITUS ON THE SKIN
EPITHELIUM OF THE RAT

EFECTOS PROGRESIVOS DE LA DIABETES MELLITUS EN LA PIEL DE RATA

Marcelo Benetti Tanga; Kemli Raquel Buttros; Cristiano Nakao & Marilena Chinali Komesu


SUMMARY: Skin serves as interface between inner aand outer environment, and acts a a mechanical barrier. Changes in its characteristics may lead to a higher pathogenicity, less defense capacity, or both. Diabetes mellitus is a heterogeneous group of chronic hyperglycemic disorders, considered a syndrome rather than a simple disease. Complications by diabetes are mostly associated with the chronicity of the disease and very little information exists about its early pathological processes. Animal models are considered very useful for the study of early diabetes. Wistar rats with lloxan induccced diabetes were used in a way to study the progresive effects of diabetes mellitus on the skin epithelium. Five animals were sacrificed at 7 days (Phase I) and five others at 26 days (Phase II) after the comprovation of hyperglycemic state. Animals not inyected with alloxan were used as control. Histometric and stereologic methods were used for the tissue analysis.

Our results allow us to observe that nevertheless progressive thickness decrease is observed with the progress of the disease, epithelial thickness is observed decreased very early in the course of the diabetes. Our results suggest that, although diabetes is a chronic disease, and many of its complications are related to the chronicity, it is reasonable to think about ddeficiency in the capacity of acting as a barier for the skin epithelium even in early phases of the disease. Professionals must be aware about patients with high risk for diabetes development.

KEY WORDS: 1. Diabetes mellitus; 2. Hyperglycemia; 3. Skin; 4. Rat.


INTRODUCTION

Diabetes mellitus is a heterogeneous group of chronic hyperglycaemic disorders. It is considered to be a syndrome rather than a simple disease. Complicated metabolic mechanisms and an increased incidence of infections are hallmarks of clinical diabetes. Several mechanisms seem to be involved, mostly associated with the chronicity of the disease. There is very little information about the early pathological processes in diabetes (Goodson & Hunt, 1979; Yagihashi, 1995 and Ptak et al., 1998).

There are no doubts that the hyperglycaemic state is a prominent characteristic of diabetes, and many complications of diabetes can be greatly influenced by the level of glycaemic control (Ruderman et al., 1992). On the other hand, the presence and severity of clinical manifestations of a number of diabetic complications can also be correlated to the magnitude of subproducts of the called advanced glycosilation (AGE=Advanced glycosilation Endproducts). AGEs are a heterogeneous group or structures, and their quantity is observed significantly increased in sera and tissues in the aged and diabetic patients (Brownlee, 1992; Lyons et al., 1992 and Yagihashi).

Animal models of diabetes are considered useful means of studying the early disease. It has been observed that alloxan destroy the beta cells in experimental animals. When injected, there is the destruction of insulin-secreting b cells within a few days, which is accompanied by typical and permanent hyperglycaemia (Mordes & Rossini, 1981; Pereira et al., 1987; Darby et al., 1997 and Ptak et al.).

Skin serves as an interface between a host's inner and outer environments, among its functions, it serves as mechanical barrier against micro-organisms and the penetration of noxious products, which means a very important protection. The antigenic load to which skin is continously exposed is high, and any changes may lead to a higher pathogenicity for external agents, less defense capacity tissue or both.

The identification and understanding of changes, whether in physiological or pathological events, are very important, and studies have not shown the implications of early phases of diabetics on the skin. The objective of our study is to monitor the progressive effect of the initial phase of experimental diabete on rat skin.

MATERIAL AND METHOD

Diabetes mellitus was induced in 180-220g male Wistar rats with an injection of 40mg/kg i.v. alloxan dissolved in physiologic saline. Treated animals were injected after 36-hour starvation. Non-injected animals were used as control. After the administration of alloxan, animals were allowed free access to food and water.

Four days after alloxan injection, the animals were tested for glycosuria by using a glucose detection tape. Those with the confirmed presence of glucose in the urine were considered diabetics,. It has been observed that diabetic animals usually survive, without any treatment, for about 10-15 days after confirmation of the hyperglycaemic state, so we kept one group (5 animals) without any treatment for 7 days after glycosuria was confirmed (11 days after alloxan injection - "Phase I" or early diabete). After this time, the other alloxan injected animals (5 animals) received anti-diabetic therapy with insulin (0.1U of insulin/100g body weight every 2 days) for 26 days ("Phase II" or chronic diabete). Five animals without alloxan injection were used as control.

All the animals (Phase I and Phase II) were sacrificed while the animals were under anesthesia, by causing a cardiopneumatic breakdown. Cardiac blood were obtained by using thoracotomy thecnique and hyperglycaemia were measured by using a Glucometer Elite (BayerTM). Skin samples were excised, fixed in 10% formalin, and processed for paraffin embedding procedures. For each specimen 6µm thick sections were made. Slides were stained with hematoxilin and eosin (H&E) for light microscopy evaluation. Morphological and morphometrical methods using a clear chamber (JENAMED) connected to a light microscope (JENA) and a Merz grid Merz, 1968) were perfomed.The data were analysed statistically using the Mann Whitney test.

RESULTS

Glycaemia evaluation: The evaluation of glycaemia showed a glucose content elevation in the blood of the animals with glycosuria (see Table I).

Table I. Glycaemia values observed on sacrifice.


Control 1 1 Days after
alloxan injection
30 Days after
alloxan injection

105 mg/dl 251 mg/dl 453 mg/dl
177 mg/d l 441 mg/dl 481 mg/dl
204 mg/dl 509 mg/dl 542 mg/dl
134 mg/dl 453 mg/dl 495 mg/dl
155 mg/dl 465 mg/dl 385 mg/dl
155 mg/dl 423 mg/dl 471 mg/dl

Test U Mann Whitney U Calc.= 0 *
P[U]= 0.004
U calc.= 0 *
P[U]= 0.004

Obs. Significant for P[U] < 0.05

Skin evaluation. Histological. The rat's skin structure is, in many aspects, similar to human skin. Both are formed by the corium or dermis (connective tissue) and epidermis (epithelium), and the tissues have the same general characteristics. In rats, as well as in humans, the specific structural caracteristics may vary depending on the region, that is, the skin thicker in some areas than in others. In the animal, the epithelial appendages are mostly hair follicle and sebaceous glands. Sweat glands are not observed in rats. The epidermis of the dorsum of a normal rat is formed by four or five cells thick with basal, spinous, granular and cornified (keratin) layers (see Figs. 1 and 2).


Fig. 1. Photomicrograph of the skin epithelium. The epithelium, connective tissue, and the presence of epithelial appendages (hair follicles and sebaceous glands) should be observed. 22,5x H.E.


Fig. 2a. Photomicrograph showing epithelial layers in control animal. The Basal, Spinous, Granulous and Keratin layers forming the epidermis should be observed. 225x. H. E.
Fig. 2b. Photomicrograph of the skin in diabetic animal, 11 days after the alloxan injection. The smaller epithelial thickness should be observed. 225x. H. E.
Fig. 2c.Photomicrograph of the skin in diabetic animal, 30 days after the alloxan injection. The difference in the epithelial thickness, mostly due to the lost of the spinous layer content should be observed. 225x. H. E.
Fig. 2. Comparison between different times after diabetes induction. Observe the progress in the alterations, and the smaller epithelial thickness early in the course of the disease (11 days after the induction).

Histometrical. Morphometric methods using Merz grid (Merz, 1968) were used to evaluate the epithelial thickness. The results, presented on Table II, confirm the histological observations, showing significant reduction in thichness of epithelium even in animals on early diabetes.

Table II. Mean values for epithelial thickness (µm) in the skin epithelium.


Evaluated structure Epithelial thickness
  Basal layer Spinour layer Keratin layer Total Epithelium
Control animals 11.15 29.19 31.88 72.22
  17.13 23.22 34.92 75.27
  10.77 21.05 26.59 58.41
  10.36 25.23 27.61 63.20
  12.55 26.55 39.57 78.67
Mean value 12.39 25.04 32.11 70.15
         
11st day after alloxan injection 7.41 18.00 32.88 58.29
  7.82 15.28 29.83 52.93
  8.34 19.49 26.45 54.28
  9.32 18.37 32.65 60.34
  9.75 15.03 31.95 56.73
Mean value 8.53 17.23 30.75 56.51
Mann Whitney test U calc.= 0 * U calc.= 0 * U calc.= 10 U calc.= 2 *
  P[U]= 0.004 P[U]= 0.004 P[U]= 0.345 P[U]= 0.016
         
30th day after alloxan injection 8.26 5.14 28.01 41.41
  5.66 4.54 26.16 36.36
  7.34 5.73 28.20 41.27
  7.20 5.50 29.35 42.05
  7.43 6.36 28.05 41.84
Mean value 7.17 5.45 27.95 40.58
         
Mann Whitney test U calc.= 0 * U calc.= 0 * U calc.= 4 * U calc.= 0 *
  P[U]= 0.004 P[U]= 0.004 P[U]= 0.048 P[U]= 0.004

Obs: Statistical significance when P[U] < 0.05.

Cariometric and stereological methods, using a clear chamber (JENAMED) connected to a light microscope (JENA), and the Merz grid (Merz) were used and allow us to perform measurement of nuclei and calculated cellular volumes. The results ar presented on Table 3 and Fig. III.

Table III. Mean values for nuclear and cellular volume (µm3) on basal and spinous layer observed on skin epithelium of studied animals.


Structure Nuclear volume Cellular volume
  Basal layer Spinous Layer Basal Layer Spinous Layer
Control animals 156.51 396.04 533.55 2227.78
  118.78 247.23 314.10 1281.69
  143.96 319.53 459.35 1388.01
  144.42 292.01 459.94 1294.52
Mean value 116.12 199.65 307.05 1664.56
  135.95 290.89 414.79 1571.31
         
11st day after alloxan injection 107.28 266.70 430.18 2906.41
  178.35 360.69 620.33 2492.41
  193.79 371.21 440.60 2491.49
  169.50 279.82 422.40 2509.40
  183.53 319.01 376.41 2673.95
Mean value 166.49 319.48 457.98 2614.76
Mann Whitney test U calc.= 5 ns U calc.= 10 ns U calc.= 12 ns U calc.= 0 *
  P[U]= 0.075 P[U]= 0.345 P[U]= 0.500 P[U]= 0.004
         
30th day after alloxan injection 102.63 151.93 88.46 341.96
  113.96 200.09 94.81 331.92
  105.01 176.78 95.28 370.32
  114.65 227.12 8.20 416.14
  100.50 202.21 92.51 349.72
Mean value 107.35 191.62 81.85 362.01
Mann Whitney test U calc.= 0 * U calc.= 3 * U calc.= 0 * U calc.= 0 *
  P[U]= 0.004 P[U]= 0.028 P[U]= 0.004 P[U]= 0.004

Obs: Statistical significance when P[U] < 0.05.


Fig. 3. Graph representing variation in nuclear and cellular volume (µm3) in skin epithelial cells during the prgression of diabetes.

DISCUSSION

Diabetes mellitus is one of the slowly evolving, chronic, noninfectious diseases. Diabetes affects cell and tissue metabolism in different ways and the precise mechanism of these alterations remained unexplained (Slavkin, 1997 and Chithra et al., 1998).

There is also little doubt that the most of the diabetic complications is associated to the prolonged exposure to hyperglycaemia, and it is also important to understand that, despite therapy and good clinical control of blood glucose levels, diabetic patients do not match the control levels achieved by individuals with normally functioning control systems (Vlassara et al., 1992; Bucala & Vlassara, 1995).

The results obtained by morphological and morphometric observation of the skin in progressive diabetes allow us to observe that: 1) There is a progressive thickness lost in the skin epithelium even in early diabetes. 2) It is very clear the deficiency in acting as a barrier with the progress of the disease, although epithelial alterations are present in the early stages of the diabetes. 3) There are indications of alteration, even in early diabetes, in cell differentiation, however, there is an evident progressive effect as observed in nuclear and cellular volumes.An important clinical implication of associated this study is the assertion of the importance of the glycaemic control in patients with diabetes mellitus, as well as the identification of epithelial alterations in the early phases of diabetes, which are possibly associated with the loss of epithelial defence capabilities.

Of course, only epidermal thickness and nuclear volumes are assessed in this study. Other end-points such as immune functions and markers in the skin will provide important supllementary information and shall be performed. However, according to these observations, it is clear the role of cronicity, the importance of time and the cumulative effects of diabetes mellitus. We can also observe some alterations present from the beginning of the diabetes, and they can also mean subclinical, but important, alterations.

Acknowledgements. We sincerely thank Prof. Dr. Miguel Angel Sala di Matteo, Faculdade de Odontologia de Ribeirão Preto/

Universidade de São Paulo, Departamento de Morfologia, Estomatologia e Fisiologia, for the castellano version of the summary.


La piel sirve como una interface entre los medios interno y externo de nuesto cuerpo. Una de sus funciones es actuar como una barrera mecánica. Cambios en sus características pueden conducir a una mayor patogenicidad y/o, a una menor capacidad de defensa. La diabetes mellitus se caracteriza por un grupo heterogéneo de alteraciones hiperglicémicas crónicas, siendo considerada un síndrome más que una simple enfermedad. Las complicaciones de la diabetes están asociadas, principalmente, con la cronicidad de la enfermedad, existiendo escasa información sobre los procesos patológicos desencadenantes. Los modelos animales son considerados muy útiles para el estudio de la diabetes inicial. Las ratas Wistar con diabetes inducida con aloxana, fueron utilizadas como modelo para estudiar los efectos progresivos de la diabetes mellitus sobre el epitelio de la piel. Cinco animales fueron sacrificados a los 7 días (Fase I) y otros cinco a los 26 días (Fase II) luego de comprobarse el estado hiperglicémico. Animales no inyectados con aloxana fueron usados como controles. Fueron usados métodos histométricos y estereológicos para el análisis de los tejidos. Nuestros resultados permitieron observar que no obstante la alteración progresiva del grosor del epitelio durante la evolución de la enfermedad, la alteración del mismo ocurre muy precozmente en el curso de la diabetes. Nuestros resultados sugieren que, a pesar de ser la diabetes una enfermedad crónica, y de que muchas de sus complicaciones están relacionadas con su cronicidad, es razonable pensar en una deficiencia en la capacidad de la piel en su acción de barrera, aún en las etapas iniciales de la enfermedad. Los profesionales debería considerar estas características en relación a aquellos pacientes con alto riesgo de desencadeanar una diabetes mellitus.

PALABRAS CLAVE: 1. Diabetes mellitus; 2. Hiperglicemia; 3. Piel; 4. Rata. 


REFERENCES

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Vlassara, H. Cell-mediated interactions of advanced glycosylation end products and the vascular wall ­ Cap 13 "in" Ruderman, N.; Williamson, J.; Brownlee, M.(Eds) Hyperglycemia, diabetes and vascular disease. Oxford University Press, New York, Oxford, 1992.         [ Links ]

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Correspondence to:
Profa. Dra. Marilena Chinali Komesu
Depto. de Morfologia, Estomatologia e Fisiologia
Faculdade de Odontologia de Ribeirão Preto ­ USP
Via do Café ­ s/n, Campus da USP ­ RP
CEP 14.040-904
Ribeirão Preto ­ SP
BRASIL

E-mail ­ mckomesu@usp.br

Received : 02-03-2003
Accepeted : 06-04-2003 


Department of Morphology, Stomatology and Physiology, Ribeirão Preto, School of Dentistry, University of São Paulo, Brazil.

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