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Journal of the Anatomical Society of India

Comparison of Toluidine Blue Vs Thionin For Mast Cells In Rat Mesentery Using Carnoy's Fixative

Author(s): Joseph, S; Das, S; Chand, R; Roopa, R. and Thomas, I. M.

Vol. 52, No. 2 (2003-07 - 2003-12)

Department of Anatomy, St. John's Medical College, Bangalore-INDIA

Abstract

Mast cells study is a useful parameter to study acute and chronic allergic conditions. It is extensively used in pharmacological studies. This study involves short term fixation of mast cells in rat mesentery with carnoy's solution comparing toluidine blue and thionin stains. It is observed that toluidine blue stains more number of cells compared to thionin though the proportion of intact, mildly degranulated and degranulated cells were the same in both the zones for the two stains. Between the two zones of mesentery studied, central zone showed more intact cells compared to peripheral zone using toluidine blue. Hence toluidine blue is preferred to stain more mast cells in rat mesentery and central zone is the preferred zone.

Key words: Toluidine Blue, Thionin, Mast Cells, Mesentry.

Introduction:

Mast cell heterogeneity was studied by Enerback (1966) in rat mesentery where in mucosal mast cells and connective tissue mast cells were distinguished. Histochemical heterogeneity of mast cells between different zones of dermis was studied by Marshall et al (1987). Rathinam et al (1990) have found differences in mast cells in rat mesentery using fresh tissue without fixation. However, since fresh tissue without fixation shows maximum degranulation and also these are not reproducible, the short fixative Carnoy's is used. The aim of the study was to find out if heterogeneity exists between different zones of rat mesentery using Carnoy's solution with respect to the well known thiazide dyes toluidine blue and thionin.

Material and Method:

Male albino wistar rats (n=7) were anaesthetized intraperitoneally with phenobarbitone sodium (40 mg/kg). The anterior abdominal wall was cut open. The proximal and distal ends of the intestine were located. Two pieces of intestine preparations with mesenteric fans were cut and transferred to a petridish containing Carnoy's solution at fixation timing of 2 hrs. The dried sections were stained using toluidine blue and thionin and observed for mast cells.

Toluidine blue method: Toluidine blue (0.1%) in formal saline (4%) was allowed on dried slide for a few seconds; the slides were then rinsed in xylene for 5-10 min. and rinsed twice with acetone. Finally, the slides were cleared in xylene and mounted in diphenylphthalein xylene.

Thionine method: Thionin (0.25%) in distilled water at an alkaline pH was used for this procedure. Slides were brought to distilled water, sequentially stained in thionin solution for 5 min, washed in distilled water for 1 min, blotted, dried and dehydrated in absolute alcohol. Finally the slides were cleared in xylene and mounted in diphenylpthalein xylene.

Mast cell counting: Ten fields were selected for counting. Five fields were from the periphery i.e. along the intestinal edge but adjacent to vascular entry route and five fields were from central zone. Mast cells could be distinguished from other connective tissue cells by the presence of cytoplasmic granules and their larger size. While counting, mast cells were divided into three types depending on the fate of the cell – intact, mildly degranulated and degranulated cells. Mildly degranulated cells were counted as a part of degranulated cells.

Results:

The data were analyzed using three way repeated measures analysis of variance (ANOVA) with three repeating measures. The three repeating measures (within subject factors) were stain (Toluidine blue Vs Thionin), zone (central Vs peripheral) and cell type (intact Vs degranulated Vs mildly degranulated). The MANOVA for repeated measures was adopted rather than the traditional mixed model approach to protect against the effects of violations of sphericity in situations where df > 1.

There was a significant main effect for stain (F = 30.81, df = 1,6, P<0.001). This indicates that overall (pooled across zones and types of cells), the total number of cells was greater with Toluidine blue than with Thionin.

There was a significant main effect for zone (F = 12×82, df 1, 6, P = 0×012). This indicates that overall (pooled across stains and types of cells) the total number of cells was greater in central than in peripheral zones.

There was a significant main effect for cells (Pillai's criterion = 0.97, F= 86.37, df = 2,5, P < 0.001). This indicates that pooled across stains and zones) the number of intact cells was significantly larger than the number of DG cells or MDG cells.

The stain by cell interaction was significant, (Pillai's criterion = 0.81, F = 10.50, df = 2,5, P = 0.016). This indicates that the total number of DG and MDG cells was proportionately the same with both stains but toluidine blue resulted in a significantly larger number of intact cells.

Table 1: Average number of intact, degranulated and mildly degranulated cells per HPF in Central and Peripheral Zones (n = 7/ cell)

Stain Zone Mean + SD
Intact DG MD
TB Central 10.4 ± 2.3 1.0 ± 0.9 0.9 ± 0.7
  Peripheral 9.4 ± 1.8 0.6 ± 0.5 0.7 ± 0.6
Thionin Central 4.5 ± 2.1 1.2 ± 0.5 0.8 ± 0.5
  Peripheral 3.8 ± 1.9 1.0 ± 0.8 0.7 ± 0.5

 

Table 2: Average percentage of intact cells per high power field

Stain Central Zone Peripheral Zone
TB 84.25 ± 10.89 87.34 ± 7.15
Thionin 66.24 ± 14.12 68.57 ± 15.63

Table 3: Average percentage of degranulated cells per high power field.

Stain Central Zone
Mean ± SD
Peripheral Zone
Mean ± SD
TB 8.89 ± 7.96 5.83 ± 4.59
Thionin 20.78 ± 10.16 17.63 ± 13.79

Table 4: Average Percentage of MD cells per HPF.

Stain Central Zone
Mean ± SD
Peripheral Zone
Mean ± SD
TB 6.86 ± 5.81 6.83 ± 5.99
Thionin 12.98 ± 8.54 13.79 ± 11.52

Discussion:

Rapid fixation is essential for best results. Hence Carnoy's solution is used at 2 hours instead of the frequently used formalin where in the fixation time is longer. Rathinam et al (1990) have found differences in cell count between peripheral and central zones using fresh tissue. In our study it was found that the total number of cells was greater in the central zone compared to the peripheral zone.

Between the two thiazide stains, toluidine blue showed more intact cells compared to thionin. Hence Using Carnoy's fixative toluidine blue is a better stain compared to thionin as more intact number of cells are seen. Failure to demonstrate mast cells in fixed tissue may be due to either dissolution of nonprecipitated glycosaminoglycans or to blocking of polyanions by cationic proteins in thionin compared to the two dyes.

This study is applicable where degranulation or an intact cell is required to be studied. The above study is of paramount importance in the study of drug applicability on mast cells.

Maximum intact mast cells were seen when:

  1. Toluidine blue stain was chosen in contrast to thionin and
  2. In central zone of rat mesentery compared to peripheral zone.

Hence the above two are recommended while studying drug trials on mast cells in rat mesentery.

References:

  1. Cooke, H.C. (1961) : A modified thionin technique for mast cells in tissue sections. Journal of Medical laboratory. 18 : 188.
  2. Enerback, L. (1966) : Mast cells in rat gastrointestinal mucosa. 1. Effects of fixation. Acta Pathology Microbiology Scandinavia. 66 : 303-312.
  3. Marshall, J.S.; Ford, G.P.: Bell, E.B. (1987) : Formalin sensitivity and differential staining of mast cells in human dermis. British Journal of Dermatology. 117 : 29-36.
  4. Rathinam, K; Mohanan, P.V.; Michael, L. (1990): In vitro Stain procedure using rat mesenteric mast cells for toxicity screening. Indian Journal of Pharmacology. 22 : 216-219.

Missing Image

Arrow shows an intact mast cell in rat mesentery under high power field using Carnoy's fixative and Thionin stain

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