Histochemical detection and comparison of apoptotic cells in the gingival epithelium using hematoxylin and eosin and methyl green-pyronin: A pilot study.

BACKGROUND: Apoptosis plays a critical role in the regulation of inflammation and host immune response. It helps in tissue homeostasis and a disturbance in this is often associated with disease. The use of histochemical stains like hematoxylin and eosin (H and E) and methyl green-pyronin (MGP) can provide a simple and cost-effective method for the detection of apoptotic cells. AIM: Study intended to analyze the expression of apoptosis in the gingival epithelium of healthy subjects and in patients with chronic periodontitis, using H and E and MGP. It is also proposed to correlate the apoptotic index (AI) of healthy individuals and those with chronic periodontitis.
MATERIALS AND METHODS: Twenty gingival biopsies were harvested from which ten samples were of healthy subjects and ten subjects who suffered from chronic periodontitis. Apoptotic cells were analyzed using MGP and H and E under light microscopy.
RESULTS: Apoptotic cells were identified at ×100 magnification and AI was calculated. Apoptotic cells were easily distinguishable in MGP stained sections when compared to those stained using H and E. Moreover, apoptotic cell count was higher in chronic periodontitis. Statistical analyses were done by Tukey's multiple post hoc procedure.
CONCLUSION: The study reveals that MGP staining can be used in a routine basic laboratory set up as one of the cost-effective methods for the detection of apoptotic cells.

INTRODUCTION

Periodontitis is a chronic disease characterized by the interaction between mixed anaerobic Gram-negative bacteria and the host inflammatory response which results in tissue destruction and tooth loss.[12]

Balancing a host-bacterium relationship is imperative for the preservation of periodontal health.[3] This equilibrium is possible through programmed cell death in concert with cell division, which generates the correct number and types of cells and maintains the balance between various cell population in organisms.[4]

Cell death is an integral part of the tissue dynamics which are mediated through two processes apoptosis and necrosis. Apoptosis is an important mechanism that is involved in maintaining the normal homeostasis not only in the evolution of life forms but also in development.

Programmed cell death also known as “apoptosis” (to fall away from) was coined by Reed to describe the unique morphology associated with a cell death that differs from necrosis.[5] It was Flemming (1885) who first described the morphological description of apoptosis. In the 1970s and 1980s, studies revealed that apoptosis not only had specific morphological characteristics but is also tightly regulated process with specific biochemical characteristics.[6] Apoptosis is considered as a genetically regulated process activated by a variety of stress stimuli, including physical and mechanical ones.

Apoptosis plays an important role in the pathogenesis of the periodontal disease. This form of cell death is important in both the inflammatory cells as well as cells of the periodontium.[7] The expression of apoptosis in connective tissue is studied at length,[8] but fewer studies are done in gingival epithelium which advocates research in this zone.[910]

Commonly used techniques for detection of apoptotic cells are terminal deoxynucleotidyltransferase (TUNEL) method, cytofluorometric assay, and DNA diffusion method, all having certain limitations such as being time-consuming, technique sensitive, and expensive.[11] On the contrary, the use of histochemical stains like hematoxylin and eosin (H and E) and methyl green-pyronin (MGP) can provide a simple and cost-effective method for the detection of apoptotic cells in a routine laboratory set up.

The present study is intended to view the expression of apoptosis in the gingival epithelium of healthy subjects and in patients with chronic periodontitis, using H and E and MGP. Moreover, proposed to correlate the apoptotic index (AI) of healthy individuals and those with chronic periodontitis.

MATERIALS AND METHODS

A total of twenty individuals were considered for the study which included ten healthy and ten chronic periodontitis patients. The study was commenced after obtaining the Institutional Ethical Committee approval. The nature and purpose of the study were explained to the subjects and written informed consent was taken. On the day of specimen collection, the periodontal parameters like bleeding index,[12] plaque index,[13] gingival index,[13] probing depth, and attachment loss were recorded using a UNC 15 periodontal probe. Periodontally, healthy group constituted of individuals with no signs of gingival inflammation, absence of bleeding on probing, probing depth < 3 mm, and no clinical attachment loss. The periodontitis group showed the presence of gingival inflammation, probing depth ≥5 mm, clinical attachment loss >l mm.

Patients with diabetes and other systemic illness, tobacco habitures, patients on any type of medication, pregnant and lactating mothers, and patients who have undergone periodontal treatment within a period of 3 months were excluded from the study.

Method of specimen collection

The gingival tissue biopsy specimen was harvested from the tooth indicated for extraction. The teeth in the healthy group were extracted for an orthodontic reason. The gingival tissue was harvested from the buccal or lingual site. The tissue was fixed in 4% neutral buffered formalin and transported to the laboratory where the specimens were routinely processed for histopathologic analyses. From the paraffin embedded tissue, two sections of 4 µm thickness were taken on microslides and one section each was stained for H and E and MGP. Routine procedure For H and E staining was conceded.[14]

Procedure for MGP staining: Methyl green-pyronin staining is a specific method for selectively staining nucleic acids. Methyl green binds specifically to DNA that is nuclei and gives green color, while pyronin is specific for ribonucleic acid (RNA), staining nucleoli red. This method has the potential to differentiate DNA and RNA. Thus, this property can be utilized for easy detection of apoptotic cells.[15]

Preparation and staining for MGP were followed as per Sigma-Aldrich specifications.[14]

Visualization of apoptotic cells in gingival epithelium was done under a light microscope at × 100 magnification and was identified based on morphological features described by Kerr et al.[16]

Both H and E and MGP stained sections were evaluated under light microscopy [Figures 1–6]. Apoptotic cells were counted in 10 random fields. In each section, 1000 cells were evaluated for the presence of apoptotic cells, and AI was calculated as the number of apoptotic cells expressed as a percentage of the total number of nonapoptotic cells counted in each case.[17]

Figure 1

Photomicrography showing gingival tissue stained with H and E Stain no (5034 & 39312)

Figure 6

Apoptotic cells indicated by arrows in gingival tissue of chronic periodontitis by methyl green-pyronin, respectively (×100)

Photomicrography showing gingival tissue stained with methyl green-pyronin Stain no (42590 & 45005)

Apoptotic cells indicated by arrows in healthy group stained by H and E, respectively (×100)

Apoptotic cells indicated by arrows in gingival tissue of chronic periodontitis stained by H and E, respectively (×100)

Apoptotic cells indicated by arrows in healthy group stained by methyl green-pyronin, respectively (×100)

AI of both the sections was compared and statistical analysis was done using Tukey's multiple post hoc procedures.

RESULTS

The apoptotic cells of gingival epithelium showed certain well-defined features which included cell shrinkage, condensation and deep eosinophilia of the cytoplasm and pyknotic, round to crescentic or irregular nucleus. Apoptotic cells were clearly tinted with dense methyl green-staining of pyknotic nuclei and dense red pyronin staining in the cytoplasm.[18]

AI was evaluated in a total of twenty cases, including ten cases each of chronic periodontitis and healthy samples. The mean AI was progressively increased in chronic periodontitis as compared to healthy gingival [Table 1]. Apoptotic cells were easily distinguishable in MGP stained sections when compared to those stained using H and E and this difference was statistically significant (P < 0.05) between the groups [Table 2 and Graph 1]. Apoptotic cell count in gingival epithelium was higher in chronic periodontitis group compared to the healthy group. When the comparison between two groups for MGP stain was made, a difference in mean number of apoptotic cells was observed but it was not statistically significant (P = 0.2).

Table 1

Mean and standard deviation of scores of hematoxylin and eosin and methyl green-pyronin stains in chronic periodontitis and healthy group

Table 2

Pair-wise comparison of chronic periodontitis and healthy groups with two stains hematoxylin and eosin and methyl green-pyronin by Tukey's multiple post hoc procedures

Graph 1

The number of apoptotic cells in healthy and chronic periodontitis group using H and E and MGP staining. H and E – Hematoxylin and eosin; MGP – Methyl green-pyronin; CP – Chronic periodontitis

DISCUSSION

Periodontitis is a multifactorial disease characterized by constant interaction between pathogenic bacteria and host defense mechanisms, which eventually leads to a destruction of the periodontium.[19]

Apoptosis can be observed under both physiological and pathological conditions and plays an important role in the process of morphogenesis and homeostasis.[5] The apoptotic cells shrinking and losing their cell-cell junctions results in detachment from the adjacent cells. Cells undergoing apoptosis are recognized by condensation of chromatin, the degradation of DNA into oligonucleosome-sized fragments, and the formation of plasma and nuclear membrane blebs. Eventually, the cell breaks apart to form so-called apoptotic bodies.[20]

Electron microscopy provides the most precise data to study cell morphology and hence is one of the most accurate methods to analyze apoptosis in tissue. Various other advanced and superior methods for detection of apoptotic bodies such as flow cytometry, electrophoresis, in situ end labeling of fragmented DNA, and TUNEL technique are at hand. But these techniques have their own disadvantages as they are expensive, needs a well-equipped laboratory, and are technique sensitive.[11] On the other hand, the histochemical technique is more cost effective, less time consuming, and is utilized in detection of apoptotic bodies. H and E are considered as a gold standard stain but due to its few drawbacks such as chromatic differentiation limits us for the identification of cells.[14] To overcome these drawbacks, various other histochemical stains such as MGP, acridine orange are in use. MGP has an added advantage of staining DNA green and RNA red and helps to visualized apoptotic cell very easily under light microscopy. Thus, the aim of the present study was to identify the apoptotic cells in gingival samples of chronic periodontitis and healthy periodontium using MGP stain and to correlate the AI in healthy individuals with those of chronic periodontitis.

In the current study, gingival biopsies of chronic periodontitis and healthy individuals were examined under the light microscopy. We demonstrated the presence of apoptotic cells by light microscopy in the epithelium of biopsy specimen from sites with probing depth ≥5 mm and attachment loss ≥3 mm.

Our findings showed that apoptotic cells are higher in biopsies from periodontitis subjects than in healthy tissue and were preferentially found in the inflammatory infiltrate.

Our observations are in accordance with the study done by Tonetti et al. and Jarnbring et al. where they found that apoptotic cells constitute less than the total cells in the gingival tissue of chronic periodontitis.[910]

A study by Tonetti et al.[9] demonstrated apoptotic cells in particularly high numbers in subepithelial clumps of inflammatory infiltrates. Cells were sturdily present in the suprabasal layer of the junctional epithelium, in close immediacy of bacterial attack. This is mainly due to the interaction between specific bacteria present in dental plaque and junctional epithelium keratinocyte. As we know, gingival keratinocytes play an essential role in the homeostasis of this epithelium and is mainly due to induction and prevention of programmed cell death. The current study we restricted ourselves to the expression of apoptotic cells in gingival epithelium as whole since there are few studies done on detection of apoptosis in gingival epithelium.[910]

Jarnbring et al.[10] studied apoptotic cells in periodontitis and gingivitis subjects in gingival tissue samples using p53 immunocytochemical technique. They found that AI was higher in periodontitis group in the most apical area in the dentoepithelium zone than in oral zone which is similar to our findings.[4]

Gamonal et al.[21] studied apoptotic events in the gingival tissue of chronic adult periodontitis patient using electron microscopy and performed immunohistochemical analysis to detect DNA fragmentation. They concluded in agreement with the present study that apoptosis is induced in the periodontal tissue by the host and microbial factors and support the hypothesis that apoptotic mechanisms could be implicated in the inflammatory process associated with gingival tissue destruction in periodontitis patients.

Kaplan [8] compared tissue expression of anti-apoptotic protein and pro-apoptotic proteins in gingival tissues of chronic periodontitis with that of healthy tissue. They concluded that in chronic periodontitis significant alterations developed in the expression of these proteins which are indicators of apoptosis.

Taking into consideration prior studies which were performed with more advanced and sophisticated methods, we found a good correlation of apoptotic cells in patients with periodontitis and healthy gingival tissue by using a simple histochemical technique.

CONCLUSION

Apoptotic cells were easily discernible in MGP stained sections when compared to H and E. It was as well seen in the current study that the AI was higher in chronic periodontitis as compared to healthy individuals. It can be stated based on the fact of the present study that MGP staining can be used routinely, it being cost-effective and a simple method for detection of apoptotic cells in a routine laboratory set up. Other tests tend to be expensive and technique sensitive. Through this trial, we hope to open avenues for researching more on simple, cost-effective methods of detecting apoptotic cells.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.