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Histological Evaluation of the Thymocyte Composition in the Thymus Gland of Mice by Using Soft Laser

Ahmed Anwar Albir

Department of Basic Sciences, College of Dentistry, University of Baghdad, Baghdad, Iraq.

Author E-mail: [email protected]

Abstract
Normal male (33) of Swiss albino mice at 9 weeks of age and body weight 41-49gm were the targets for this experimental study. These targeted mice were divided into three targeted groups (n=11each). The first targeted group of mice was kept as unirradiated control. Whereas both second and third targeted groups of mice were used as irradiated groups with laser. Our presented work was considered as a suggestion that established possible influences of soft laser at the cellular structure of the common thymus gland in mice. The outcomes of our work demonstrate the supposition and the laser caused marked changes in the cellular consists of the thymus gland particularly the consist of the thymocytes which included raised volume of the thymocytes and reduplication of the cellular nuclei. These alterations were numerous in the contains of the thymus gland thymocytes of the second group of mice irradiated with laser, but the mentioned alterations were more numerous in the structure of the thymocytes of the thymus glands to the third group of mice irradiated with laser.

Key Words: Medical Laser, Thymocytes of the thymus gland, Histological alterations, Mice.

Introduction
Usually the location of thymus in the mediastinum (1, 2). It is build from twice lobes that separated into many lobules; each one is split up from the other by trabeculae that build from connective tissue. Every lobule consists of two main components, an external cortex and an internal medulla (3). The cortex and the medulla meet at region called corticomedullary junction (CMJ) which is considered as the tip of entrance and the way out to develop thymocytes (4). T cell precursors come into presence in the bone marrow and circulating to break the thymus by the corticomedullary cross, and through the intra-thymic trip, T cells usually termed thymocytes (5-8). In thymus and throughout the development, growing thymocytes shift from cortex to medulla (9-11). It shouldn’t be beglect the generating of T cells by thymus through the life span (12, 13). In addition to the greater composition of thymocytes, the thymus microenvironment has different kinds of thymic epithelial cells (TEC), stroma cells, macrophages, fibroblasts and dendritic cells. All these cells contribute in thymocyte maturation process (3).
Corpuscles thymic medulla (Hassall) has different dimensions and contains large sum of thymic epithelial cells (TECs). Those cells secrete many cytokines that are significant for the control of the activity of dendritic cells as well as the differentials of approval T cells (2).
Laser phototherapy has a main role in the medical fields including: promoting wound healing, tissue repair and prohibition tissue from death, relief of inflammation in chronic diseases and injuries with its associated pain and edema, relief of neurogenic pain and some neurological problems (14).
The purpose of this experimental study was to address the question of whether the soft laser could affect the cellular structure of the thymus gland particularly thymocyte. In reality, the presented work is a hypothesis needs to be proved.

Statistical factors
Mean: The average pixel value taken to be equal to the average brightness or intensity and computed using equation (1), 18:
(1)

Where M, N are the dimensions of image and I(x,y) is the pixel intensity value (0-255 for a 256 bit image).

Variance: Coefficient of variance is defined as the ratio of standard deviation to the mean and calculated from following equation 18:

(2)
Sometime Cv is assumed as a measure to difference in digital image.

Standard deviation (Std): The Standard Deviation is the most commonly used index of variability and is a measure related to the average distance of the scores from their mean value. This is also an indicator of contrast in the image. It is computed using the following result 18:

(3)
The standard deviation is important in identifying the `details content in an image.

Materials ; Methods
33 normal male of Swiss albino mice at 9 weeks of age and body weight 41-49gm were selected to be the goals for this empirical research, and this number of mice that could be sufficient was chosen in order to ensure that our presented work will achieve better results. This mentioned number of mice was divided into three targeted groups (n=11 each). The first targeted group of mice was kept as unirradiated control in order to compare the results of other groups of mice irradiated with laser with that of the unirradiated control group of mice. Both second and third targeted groups of mice that constitute irradiated groups with laser were anaesthetized and exposed to a low power gallium aluminum arsenide laser (Ga Al As) of wavelength (lambda = 830nm). The beam of mentioned laser was directed above the heart (location of the thymus gland) of the targeted mice and the object was one centimeter distant from the laser source. Once daily irradiation with laser was performed for 30 and 35 minutes to the targeted mice which included both second and third irradiated groups respectively.
The entire period of experimentation (laser irradiation) lasted 12 consecutive days. Both time of receiving laser irradiation and entire period of experimentation (laser irradiation) were arranged for each irradiated group of mice as in the following table:

After the end of the entire period of experimentation (laser irradiation), all the mice including both normal control group and the other irradiated groups were killed and their thymus glands were rapidly obtained. Sections of thymus glands were prepared by using a routine procedure aiming at studying the histological evaluation of the thymocyte composition by light microscopy. Photographs were made at original magnification.

Results
The findings of our experimental work confirmed the existence of marked effects of soft laser on the thymocytes structure. These marked effects comprehend as arranged in Table1, Table 2 and Table 3.

Discussion
The current experimental study perform a hypothesis that established probable effects of laser irradiation when utilized to a normal tissue like the thymus gland structure of mice. So, our feedback proved this hypothesis and the laser caused notable changes in the cellular structure of the thymus gland particularly the thymocyte structure.
In the subsequent group of mice irradiated with laser for 30 minutes once daily during the entire period of experimentation (12 consecutive days), the laser was effective and result in structure changes of the thymocytes of the thymus glands which were abundant and inclusively rise the volume of the thymocytes and duplication of the cellular nuclei (Image 2) analogy with the thymocytes glands structures. The first unirradiated (control group) of mice which have normal composition (Image1). while in the third group of mice irradiated with laser for 35 minutes once daily during the entire period of experimentation (12 sequential days), the laser was more effective and in turn, caused changes in the composition of the thymocytes of the thymus glands which were more abundant and included enlarged size of the thymocytes and duplication of the cellular nuclei (Image 3) compared with the composition of the thymocytes of the thymus glands of the second group of mice irradiated with laser (Image2). It could be said that these alterations that occur in the composition of the thymocytes were due to the following:
1. The selective laser of the presented work was suitable for stimulation of the cellular composition of the thymus gland particularly stimulation of the thymocytes components

2. The steady increasing in times of our doses of laser irradiation were appropriated because these mentioned times gave the laser the opportunity to alter the thymocytes components.

3. The total period time of experimentation (laser irradiation) of the presented work which lasted 12 consecutive days was sufficient to achieve noticeable results.

We may offer possible explanations for the alterations induced by soft laser through its effects on cellular component of the thymus gland especially the component of the thymocytes. However, in the same time, our results suggest a gain in functional activity termed a successful stimulation of the soft laser to the cellular component of the thymus gland particularly stimulation of the composition of the thymocytes.

In another words, soft laser examined the efficient ability of the thymus gland by altering the component of the thymocytes using regular times of receiving laser irradiation. Laser can stimulate cellular functions by applying it at an appropriate dose (15, 16).

However, the principles of laser action in each cell and tissues are still not well known (17).
As seen in table 3 and figure 1 the statistical features (mean, variance, std) of R, G and B bands of 3 images before and after different time of receiving laser irradiation, show no much different values. So the images preserve their main characteristics except the alterations resulted from stimulating state of laser irradiation (18).
Finally, many questions remain unanswered about the mechanism of laser action and its effects on diverse sorts of cells and tissues. These inquires should be answered through time and careful investigation.

Conclusive Remarks:
• Soft laser was a successful biostimulative tool in proving our hypothesis and reflect to the changes the hymocyte structure which has raised the cell dimensions as well as duplication of the cellular nucleus.
• Low energy laser, and as a consequence of allowing for biostimulatory effects will reinforce the functional immune reactions of the thymus gland and that, in turn, will quicken the treatment of dangerous diseases especially cancerous diseases.

Conflict
No conflict of interest

References
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8. Godfrey DI, Kennedy J, Mombaerts P, Tonegawa S, Zlotnik A. 1994. Onset of TCR-beta gene rearrangement and role of TCR-beta expression during CD3-CD4-CD8- thymocyte differentiation. J. Immunol. 152(10), 4783-92.
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14. Hamblin MR. Waynant RW. 2006. “Mechanisms for Low-Light Therapy.” Proc. of SPIE 6140 614001-614010.
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16. Gomez-Villamandos RJ. Santisteban-Valenzuela JM. Ruiz-Calatrava I. Gomez-Villamandos JC. Avila-Jurado I. 1995. He-Ne laser therapy by fibroendoscopy in the mucosa of the equine upper airway. Lasers Surg. Med. 16, 184-8.
17. Rocha-Junior AM. Vieira BJ. De-Andrade LCF. Aarestrup FM. 2007. Effects of low-level laser therapy on the progress of wound healing in humans: the contribution of in vitro and in vivo experimental studies. J. Vasc. Bras. 6, 258-266.
18. Gomes J. Velho L. 1997. Image Processing for Computer Graphics, Silvio Levy, New York, Inc.

Table 1: Showing Both Time of Receiving Laser Irradiation and Entire Period of Experimentation (laser irradiation) for each Irradiated Group of Mice
Group Number of mice Time of receiving laser irradiation Entire period of
experimentation
(laser irradiation)
First
(unirradiated control) 11 ________ ________
Second
(irradiated with laser) 11 30 minutes once daily 12 days
Third
(irradiated with laser) 11 35 minutes once daily 12 days

Table 2: Representing the Histological Evaluation of the Effects of Soft Laser on the Thymocyte Composition
Number of mice per group = 11
Name of the group Time of receiving laser irradiation Entire period of
experimentation
(laser irradiation) Histological evaluation of the composition of the thymocyte
First
(unirradiated control) ________ ________ Normal composition of the thymocytes (Image 1)
Second
(irradiated with laser) 30 minutes once daily 12 days Increased size of the thymocytes, and multiplication of the cellular nuclei were clearly marked (Image 2)
Third
(irradiated with laser) 35 minutes once daily 12 days Increased size of the thymocytes, and multiplication of the cellular nuclei were more clearly marked (Image 3)compared with the second group of mice irradiated with laser (Image 2)

Table 3: Mean, Std and Variance values of the three images of their R, G and B bands.

Image No. Bands Mean Stdev Variance

Image 1 R 165.150983 46.764264 71.8267061873
G 108.742503 58.336759 43.8720096846
B 157.461023 37.617848 43.8720096846

Image 2 R 171.112174 54.092200 50.5496214409
G 118.491401 74.016735 49.6635284603
B 158.950830 47.340910 28.4631612353

Image3 R 163.338496 36.398892 22.4798948187
G 93.329292 53.928686 28.9758167776
B 141.079776 35.272071 16.4025370093

Image 1: Histology of the lobule of the thymus gland of the first unirradiated control group of mice showing normal composition of the thymocytes. H & E, 40 × Magnification.

Image 2: Histology of the lobule of the thymus gland of the second group of mice irradiated with laser showing abundant alterations in the composition of the thymocytes included increased size of the thymocytes and multiplication of the cellular nuclei. H & E, 40 × Magnification.

Image 3: Histology of the lobule of the thymus gland of the third group of mice irradiated with laser showing more abundant alterations in the composition of the thymocytes included increased size of the thymocytes and multiplication of the cellular nuclei. H & E, 40 × Magnification.

Figure 1: Column chart of statistical values Mean, Std and Variance of R, G and B for each image.

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