Jiri Luza, Department of Physiology, Palacky`s University, Olomouc, CZ
Jiri Hubacek, E.N.T. Department, Palacky`s University, Olomouc, CZ

 

ABSTRACT

The aim of this study is to evaluate in vitro the effect of Helium-Neon (He-Ne) laser irradiation on the viability, adherence, phagocytic activity of the polymorphonuclears and monocytes. Also the level of metabolic processes in phagocytizing blood cells monocytes and polymorphonuclears was estimated and evaluated by the INT-test. For evaluation of the leukocyte adherence, the method of MacGregor was used. Phagocytic activity was examined by classical method using microspherical hydrophylic particles (Hema-particles). He-Ne laser in a small dosage (< 0.8 J) increases the leukocyte adherence, after higher irradiation dosage (> 1.2 J) the leukocyte adherence is decreased. The laser effect on phagocytic activity of both types of blood cells, polymorphonuclears and monocytes is similar. Small dosage of laser radiation increases phagocytic activity, and after higher laser irradiation phagocytic activity is decreased. Also the changes of the level of metabolic processes in phagocytizing cells are very similar with the changes of phagocytic activity. The viability of the white blood cells examined after higher laser irradiation is gradually decreased.

 

INTRODUCTION

Phagocytic cells, polymorphonuclears and monocytes play key role in nonspecific immunity. From this point of view the influence of laser irradiation has been studied. There is much information on relationship between laser irradiation and reaction of the immune system in literature. As this information is often contradicting, the aim of this study is to evaluate in vitro the effect of He-Ne laser irradiation on viability, adherence, and phagocytic activity of polymorphonuclears and monocytes, and also to evaluate the level of metabolic processes in phagocytizing blood cells.

 

METHOD

The experiment was performed with 240 blood samples from 10 rabbits, silver-grey cross breeds of both sexes and the mean weight of 3600 + 450 g.

Blood samples were taken from vena marginalis. As an anticoagulant Heparin Spofa (15 units/ml blood) was used.

Cells viability was studied by a simple method according to Hanks and Wallace, based on different permeability of cell membrane for trypan blue in live and dead cells.

For evaluation of leukocyte adherence the method of MacGregor, modified by Cates, was used. The noncoagulated blood is injected into glass tubes (Pasteur pipettes) of 5 mm in internal diameter and is left to flow at room temperature over 15 mm column of nylon fibres weighing 50 mg (LP-1 Leuko-Pak Leukocyte Filter). Each blood sample is evaluated in three tubes. Adherence is determined by comparison of the number of leukocytes in blood passed through the nylon adherence column with that found in blood before the experiment. Total number of leukocytes and differential blood counts are evaluated.

Phagocytic activity of leukocytes was determined by a classic routine method of substracting the phagocytic cells from the blood smear. Microspherical hydrophilic particles were used for an objective testing of phagocytic activity.

Level of metabolic processes in phagocytizing blood cells was estimated by the INT-test which evaluates tetrazolium - reductase activity of the phagocytes. The INT-test detects indirectly some metabolic events occuring in phagocytizing cells and associated with phagocytized corpuscle material (zymosane). Tetrazolium salt is an artificial acceptor of hydrogen (which is released fromNADH via NADH-oxidase) and is reduced into colored formazane. The amount of originated formazane, i. e. the rate of reductase activity of leukocytes, is estimated photometrically (485 nm).

All of the above mentioned functional parameters of leukocytes were estimated before laser irradiation (control group), and after laser irradiation. The times of laser irradiation exposures were 5 s (0.2 J), 10 s (0.4 J), 20 s (0.8 J), 30 s (1.2 J), and 60 s (2.4 J).

For our irradiation a He-Ne laser with 40 mW output was used.

All the results obtained were subjected to statistical evaluation (unpaired Student`s test was used).

 

RESULTS

The study of leukocyte adherence was carried out as it is described in the methodological section. The test was done on samples of irradiated and non-irradiated blood cells. Leukocyte adherence after laser irradiation was changed. Leukocyte adherence after 5, 10, 20, and 30 seconds of laser irradiation was increased (about 15.9%), but all these changes were on the border of statistical significance. The decreasing of leukocyte adherence after 60 seconds of laser irradiation was statistically significant (p < 0.02). (Figure 1).

Fig. 1 - Leukocyte adherence
K = total leukocyte count of the peripheral blood of intact rabbits. Leukocyte count of blood passed through the adherence nylon column, k = before laser irradiation, 5 s (0.2 J), 10 s (0.4 J), 20 s (0.8 J), 30 s 1.2 J), 60 s (2.4 J) = after time of laser irradiation. Leukocyte adherence after 5, 10, 20 and 30 s of laser irradiation was increased (15.9 %) but all these changes were on the border of statistical significance.

Adherence of polymorphonuclears was significantly increased after 20 seconds of laser irradiation by 25.9% (p < 0.02). Adherence of PMN`s after 60 seconds of laser irradiation was decreased on the level of adherence of the PMN`s of the control group (Fig. 2).

Fig. 2 - Polymorphonuclears (PMN) adherence
K = total PMN`s count of the peripheral blood of intact rabbits. PMN`s count of blood passed through the adherence nylon column, k = before laser irradiation, 5 s (0.2 J), 10 s (0.4 J), 20 s (0.8 J), 30 s 1.2 J), 60 s (2.4 J) = after time of laser irradiation. Adherence of PMN`s was significantly increased 20 s of laser irradiation (25.9 %) (p < 0.02).

Laser irradiation did not essentially influence the adherence of lymphocytes (Fig. 3).

Fig. 3 - Lymphocyte adherence
K = total lymphocyte count of the peripheral blood of intact rabbits. Lymphocyte count of blood passed through the adherence nylon column, k = before laser irradiation, 5 s (0.2 J), 10 s (0.4 J), 20 s (0.8 J), 30 s 1.2 J), 60 s (2.4 J) = after time of laser irradiation. Laser irradiation did not essentially influence the adherence of lymphocytes.

He-Ne laser irradiation influenced phagocytic activity of the PMN`s and MO`s, too. Low doses of laser irradiation increased phagocytic activity in both types of leukocytes. Maximum increase of phagocytic activity of the PMN`s could be measured after 5 seconds of irradiation, phagocytic activity of the PMN`s was significantly (p < 0.05) increased by 10.8%. (Fig. 5).

Phagocytic activity of the monocytes after low doses of laser irradiation was increased more than the phagocytic activity of the PMN`s, by 22 % (p < 0.02). Extending the irradiation period step by step decreased stimulating effect of laser irradiation on phagocytic activity of the PMN`s and MO`s, too. Phagocytic activity of both the phagocytizing cells, MO`s and PMN`s, after 60 seconds of laser irradiation was decreased on the level of the phagocytic activity of the control group (Fig. 4).

Fig. 4 - Phagocytic activity of monocytes in vitro
K = phagocytosis before laser irradiation, 5 s (0.2 J), 10 s (0.4 J), 20 s (0.8 J), 30 s 1.2 J), 60 s (2.4 J) = time of laser irradiation. Phagocytic activity of MO`s after low doses of laser irradiation was increased more than 22 % (p < 0.02).

Fig. 5 - Phagocytic activity of PMN`s in vitro
PMN`s phagocytosis (%). K = phagocytosis before laser irradiation, 5 s (0.2 J), 10 s (0.4 J), 20 s (0.8 J), 30 s 1.2 J), 60 s (2.4 J) = after time of laser irradiation. The most significant increase of phagocytic activity of PMN`s was measured after 5 s of irradiation (10.8 %, p < 0.05).

Both the functional leukocyte parameters which were studied, leukocyte adherence and phagocytic activity of the leukocytes were after low doses of laser irradiation increased.

In correspondence with the modification of adherence and phagocytic activity of white blood cells after laser irradiation, the INT-test detecting the changes of the metabolic activity in phagocytizing cells aslo showed a marked increase of formazane production in white blood cells which were exposed to the lower doses of laserr irradiation. The greatest increase of formazane production could be observed in blood samples which were irradiated for 20 seconds ( 137 %). These changes were onthe border of statistical significance. Increasing the doses of laser irradiation gradually decreased formazane production (Fig. 6).

Fig. 6 - Intensity of metabolic changes of phagocytizing white blood cells (detected by the INT-test, expressed in %)
K = values of the INT-test in white blood cells before laser irradiation, 5 s (0.2 J), 10 s (0.4 J), 20 s (0.8 J), 30 s 1.2 J), 60 s (2.4 J) = after time of laser irradiation. The greatest increase of formazan production could be observed in blood samples irradiated for 20 s (137 %), 5 s (0.2 J), 10 s (0.4 J), 20 s (0.8 J), 30 s 1.2 J), 60 s (2.4 J) = after time of laser irradiation. 

During evaluation of phagocytic activity by visual means we could see that many many of the leukocytes, PMN`s and MO`s were damaged. Also many forms of leukocytes, which were called blood or cell shadows by Netousek, could be observed.

For this reason we have made an evaluation of cell viability. Total count of the white blood cells in all experimental groups after laser irradiation was decreased, but after 60 seconds of irradiation the decreasing of the leukocyte viability was statistically significant (p < 0.02). (Fig. 7).

Fig. 7 - Leukocyte viability in vitro expressed in %
Left column = leukocyte viability tested immediately after laser irradiation. Right column = leukocyte viability tested 2 hours after laser irradiation. K = before laser irradiation, 5 s (0.2 J), 10 s (0.4 J), 20 s (0.8 J), 30 s 1.2 J), 60 s (2.4 J) = time of laser irradiation.

 

DISCUSSION

Evaluation of viability, adherence, phagocytic activity, and metabolic activity of the phagocytizing white blood cells after laser irradiation showed that all these functional parameters were modified. In general, we can say that exposition to lower doses of laser irradiation in vitro increased markedly adherence (recent information in literature has shown that adherence of neutrophylic granulocytes to vascular endothelium is one of very important factors influencing inflammatory reaction in all its phases; adherence of the neutrophils is an essential initial step for phagocytosis),  phagocytic activity, and metabolic activity of PMN`s and MO`s, too. Exposure to greater doses of laser irradiation (2.4 J and more) changed all these functional properties of PMN`s and MO`s; viability, adherence and phagocytic activity were significantly decreased (p < 0.02).

Information in literature explains the effect of laser irradiation on living biological subjects as a result of complex influences of both thermal and photochemical components of laser irradiation (in He-Ne laser the thermal component being absent). The terminal effect is dependent on time of exposure to the irradiation. It seems that the effect of stimulation can be connected with functional reversible changes in cells, for example the increasing of enzymatic activity, or changes of plasma membrane properties (for example plasma membrane permeability). On the other hand, inhibitory effect is connected with morphological changes which have irreversible character.

In spite of all recent literary information, it is very difficult to explain laser stimulative effect, and therapeutic use of stimulating laser irradiation has still empiric character.

Literature available with the authors.