# Morphological Changes of Gingiva in Streptozotocin  Diabetic Rats

Authors: C. Tesseromatis; A. Kotsiou; H. Parara; E. Vairaktaris; M. Tsamouri
Journal: International Journal of Dentistry
 (2009)

---

## Abstract

Gingivitis and periodontitis are chronic bacterial diseases of the underlying and surrounding tooth tissues. Diabetes mellitus is responsible for tooth deprivation both by decay and periodontal disease. The streptozotocin-induced diabetes results in a diabetic status in experimental animals similar to that observed in diabetes patients. The aim of the study was to investigate the relationship between the gingival lesions and the microangiopathy changes  in streptozotocin-induced diabetes mellitus.  Forty male Wistar rats were divided into two groups (control and experimental). Diabetes mellitus was induced by 45 mg/kg IV streptozotocin. The histological investigation of the marginal gingival and the relevant gingival papilla showed inflammation of the lamina propria and the squamous epithelium as well as marked thickness of the arteriole in the diabetic group, but no changes were observed in the control group. The results suggested a probable application of a routine gingival histological investigation in diabetic patients in order to control the progress of disease complications. It may be concluded that histological gingival investigation can be used as a routine assay for the control of the diabetic disease and prevention of its complications.


---

## Body

## 1. Introduction

Gingivitis and periodontitis are chronic
bacterial diseases of the underlying and surrounding tooth tissues. The initial
factor in the development of the periodontal disease is the host response and
its defense capacity to the microbial stimuli. Systemic factors modify all
forms of gingivitis and periodontitis through their effect on the physiological
immune and inflammatory defense. It has been reported [1] that nerve fibers are involved in the neurogenic inflammation induced by
mechanical or chemical irritations in the gingival and the underlying tissues. It
has also been suggested that in diabetes mellitus the unmyelinated small
diameter fibers are impaired as a result of diabetic neuropathy. Furthermore,
many studies have been focused in the impairment of bone mass, occurring in
diabetes mellitus. Microangiopathy at the bone tissue was suggested as a
possible cause of diabetic osteopenia [2].
Diabetes can have an impact on the bone through multiple pathways, some with
contradictory effects, including obesity, changes in insulin levels, higher
concentrations of advanced glycation end products in collagen, hypercalciuria
associated with glycosuria, reduced renal function, lower insulin-like growth
factor-I, microangiopathy, and inflammation [3].Furthermore, diabetes
mellitus is responsible for tooth deprivation both by decay and periodontal
disease. Systemic diseases are
associated with a higher experience of caries, a high ratio of
decayed-to-present teeth, and more gingival and periodontal problems. Patients
with high blood pressure, osteoporosis or diabetes mellitus tended to have
poorer gingival or periodontal conditions, fewer teeth, and higher risk of
edentulousness [4]. In addition, it was found that a larger number of
oral streptococci adhered to the tooth surfaces are observed in nonobese diabetogenic mice that spontaneously develop
insulin-dependent diabetes mellitus [5].The aim of the study was to investigate the
relationship between the development of gingival lesions and the presence of
microangiopathy in streptozotocin-induced diabetes mellitus.


## 2. Material and Methods

Forty male Wistar rats of average body weight of 200 g were divided
into groups A (experimentaln=20) and B (control, n=10). They were housed five
per cage at a constant room temperature (22±1°C) under a 12-hour light/12-hour
dark (light period 00.8–20.00 hours)
cycle. Food and water were provided ad libitum. Animals were cared for
accordance with the principles of the “Guide for the Care and Use of Experimental
Animals”  [6]. The animals of group A were injected once IV with
streptozotocin 45 mg/kg
in the jugular interna. The duration of the experiment was 90 days. The blood glucose
levels were estimated every week with wash-off strips (Dextrostix, Ames
Division, Miles Laboratories, Rexdale, Ontario, Canada)
in blood obtained from the tail vessels. In parallel, glucose was also
qualitatively assayed in urine with urineteststrips (Glukotest Accu-test
Roche). The animals' body weight as well as their food intake was determined.
The animals were sacrificed by decapitation, and gingiva specimens obtained
from the incisor area of the mandible were washed with water and then conserved
in 10% formalin solution for further histological examination with the light
microscope. The histological slices of gingival specimens were stained with
haematoxylin-eosin by light microscope. The values were expressed as mean ± standard
deviation (m±SD). The statistical
analysis was performed by Student's t-test and by x2 analysis. P<.05 was considered as an acceptable level of
significance.


## 3. Results

The induction of diabetes mellitus was assessed the day after
streptozotocin injection by evaluating clinical symptoms such as frequent urination, increased
appetite, and weight loss. In comparison to the control group, the experimental
animals exerted a
hyperphagia accompanied with an increased daily food consumption. Furthermore,
the streptozotocin animals had increased serum glucose and increased
glycosylated haemoglobin (Hb A1c)
levels. The severity of diabetes was indicated by the
statistically significantly decreased body weight in the experimental animals
in comparison to controlsP<  ,001
(Table 1). The quantity of daily
food intake was increased in diabetic animals compared to controls. Blood
glucose levels were significantly increased in the experimental group (Table 1).
The levels of Hb A1c were lower in the control compared to the experimental group. The urination of the experimental group was frequent and the shavings
of the cages needed to be changed twice daily.Table 1
Clinical indices and laboratory findings upon diabetes mellitus induction.
Control animalsStreptozotocin-injected animalsDaily food intake (g)14,86 ± 5,0919,25 ± 0,95**Body weight (sacrifice day) g190 ± 17160 ± 20**Hb A1c (%)5,5%8,2 ± 1,4%**Serum glucose mg/dL90250****P<  ,001 versus control.The experimental animals had a mortality of 10% during the
experimental period, while all the control animals remained alive until the end
of the experimental procedure.The histological findings of the experimental group were as follows.
Through haematoxylin-eosin
stain, the biopsies from the marginal gingivae and the gingival papillae
from the incisor area showed inflammation of the lamina propria, formation of
new vessels with various wall thicknesses, and hyperaemia. This alteration was
localized in the internal site of the gingiva, which was in contact with the
tooth surface.The gingival specimens obtained from
the molar area of the mandible of the buccal and lingual sites showed moderate to
high hyperceratosis and mild inflammation. All the experimental animals showed
moderate-to-severe angiitis, which was not observed in the control animals (see
Table2, Figures 1
and 2).Table 2
Histological findings.
Margin gingival incisor areaDiabetes mellitusControlInflammation++++Neoangiogenesis++++Vessel wall thickness++++Buccal/lingal gingivaHyperkeratosis++++Epithelium hypertrophy++—Figure 1
(▴) Focal perivascular and diffuse
inflammation of the lamina propria,  (↑) neo-angiogenesis,
and (*) hyperplasia of the squamous epithelium.Figure 2
(*) Hyperplasia of the squamous
epithelium,  (▴) diffuse and perivascular inflammation of the lamina propria,
and (↑) thickening of the wall of some arterioles.Figure 3
(*) Normal gingival mucosa. normal squamous epithelium, (↑) normal propria
vasculature, and absence of inflammation.


## 4. Discussion

Recent drastic increase in diabetic population poses serious
problems in both health sciences and socioeconomic services. The recent drastic increase in the diabetic population
poses serious problems in both health sciences and socioeconomic services. The
most important issue in the clinical management of diabetic patients is the
chronic complications that contribute to a high morbidity and mortality [7, 8].Hyperglycaemia, as a common
feature of diabetes mellitus, is a cause of different pathogenic mechanisms
influencing endothelial function.Diabetes mellitus results in the development of large and
micro-vessel damage. A series of changes occur independently of the presence of
atherosclerosis. The abnormalities include accumulation of PAS-positive
material, laminin, fibronectin, type IV collagen, and connective tissue with
lack of acid mucopolysaccharides, and deposition of calcium. It is of
particular interest that accumulation of PAS-positive material and lack of acid
mucopolysaccharides are recognized as the histological markers of diabetic
microangiopathy [2, 8–12].In this study, the administration of streptozotocin in rats induced
a diabetic status in the experimental animals similar to that observed in
diabetic patients, as shown by the manifested clinical symptoms in accordance with
previously reported literature [2, 13, 14].The changes observed in bed
vasculature concerned macro- and microangiopathy. The extention of
microangiopathy is known to impair the function of various organs and systems
and common problems are manifested as retinopathy, nephropathy, and
neuropathy [15]. Periodontal disease is considered as the sixth complication of diabetes
mellitus. It has long been observed that diabetic patients have greater tooth
loss due to periodontal disease than nondiabetics of comparable age [16].
The severity of the periodontal disease is absolutely influenced by the degree
of diabetic status [17]. The
phenomenon may be due to the high tissue glucose concentration, as well as to
the presence of metabolic products of the impaired glucose metabolism and it is
recognized histologically as enlarged vessel wall with a narrowing of vessel
lumen diameter. This process induces disabilities of vessel wall and, in
general, leads to abnormal vasculature [12, 18]. Our results are in
agreement with those of other investigators, who reported narrowing of vessel
lumen diameter in diabetic subjects as observed by the increased uptake of PAS
deposition in the vessel walls [9].These changes can be aggravated through inflammatory cell infiltration that
occurs after one week of steptozotocin treatment in the gingivomucosal tissue as reported
by Fehér et al. [1].
Despite multiple and long-term studies, the pathophysiology of diabetic
microangiopathy and its pathogenesis are not fully elucidated. Under chronic
hyperglycemia, early stimuli elicit adaptive reactions of tissues showing acute
inflammatory processes of vessel walls and changes of microangiopathy. The
impaired glucose metabolism is recognized histologically as enlarged vessel
wall with a narrowing of vessel lumen diameter. This situation in dental care
can be recognized by delay healing of tooth extraction sockets, periradical lesions,
and periodontal disease [4, 19, 20].In addition, it has been suggested that the unmyelinated small-diameter
afferent nociceptive C-fibers are impaired in diabetes mellitus, which indicates that in
the streptosotocin-induced diabetic rat, gingivomucosal tissue is the prerequisite for
neurogenic inflammation induced by mechanical or chemical irritations causing a
pronounced vessel permeability. Furthermore, diabetic changes may be accompanied by
decreased collagen production in rat periodontal tissue [21].This process induces disabilities of vessel wall such as narrowed vessel
calibre and in general leads to the formation of an abnormal vasculature [12, 19].
Since the ability of the diabetic's circulation to distribute blood is
affected, especially during increased blood flow causing severe disturbances, the
surrounding and underlying tooth tissues have poor nourishment, which, in
relation to the high blood glucose levels, promotes the colonization of
overdeveloped microflora in the oral cavity [22]. The presence of PAS
material deposition in gingival vessel can be considered as an index of severe
diabetic damage [9]. Therefore, slow flow of nutrients and high
infection liability promote the destruction process of the periodondium [23].An important injury that leads to severe handicap in diabetic
patients is the development of retinopathy that usually leads to blindness [24].
Most recently, it has been proven that the risk of proliferative diabetic
retinopathy was higher in the presence of the periodontal disease [25].
In addition, the occurrence of neuropathy in long-term type 2 diabetes is
related to tooth loss and tempomandibular joint disfunction [26].Therefore, the
investigation of the surrounding oral cavity tissues in diabetic patients can
demonstrate changes or signs that may alert the physician to control or prevent
the development of diabetes mellitus. Furthermore, histological gingival
analysis may be routinely utilized for the control of the diabetic disease and
perhaps may be considered as a diagnostic method of the severity of the
disease.



---

*Source: 725628-2009-01-27.xml*