Morphological and Cytochemical Research of
Indirect Laser Rays' Influences on Rat's Liver Damaged by CCL⁴

Object and experiment’s conditions.

Liver of male white rats (140 ± 5,5g) were examined in this study. 90 rats had 9 hypodermic CCL4–injections on sunflower-seed oil (1:1) in the dose 0,12 ml on 100g of animal’s weight twice a week during 30 days [7].

Five rats’ livers were fixed for definition of morphological changes after 30 days of CCL4–injections. Other animals were separated on two groups. The first group was consisted of 45 rats. Animal livers had indirect treatment by He-Ne laser rays (632,8 nm, 20 mWatt, 3,5 mWatt/cm², 5 minutes) during 20 days. The second group of animals was control. The third group was consisted of 15 intact rats with normal livers. The rats’ livers were fixed after 3,6,12,20 He-Ne laser treatments (3, 6,12,20 days after cessation of CCL4-bringing).

Histologic Study

The tissue samples of central part of liver’s right lobe were fixed in 10% neutral form alin solution, paraffin embedded, cut in 4µm sections and stained with hematoxylin-eosin and the solution of picric acid and fuchsin [8]. Some tissue samples after fixation in formalin were cut in 20-30µm sections on freezing microtome, stained with Sudan-3 or Sudan Black for lipids-revealing [8].

Electron-microscopic Study

1mm³- tissue samples were fixed in 2% OsO4 on phosphate buffer with 4,5% sacharose (pH = 7,35) by Palade method, modified by Cauldfield [9]. Fixation was conducted at +4°C during 4 hours. After dehydratation in alcohol (increasing concentrations) and acetone the object was Epon-embedded, cut in ultra-sections [10].

The ultra-sections were placed on nets covered by collodion or formvar film, stained with uranyl acetate and lead citrate [11], examined through an electron microscope EM-100B.

Cytophotometric study

Liver smears were fixed in methanol, stained for revealing of nucleus acids, proteins, glycogen. The smears were stained with gallocyanin-chrom alum for nucleus acids revealing, with mercuric chloride solution of bromine-phenol blue for proteins, with Schiff-periodate acid with control by 1% pancreas amylase at 37°C for glycogen revealing [8,12].

The liver smears were prepared from 3-5 rats for one histochemic reaction. 75 nuclei or 75 cytoplasms from one animal were examined for one histochemic reaction.

Photomeasures of histochemical reaction’s products were conducted with cytophotometre worked in spectrum's visible part.

Optical density C was defined:

where J1 was a value of light stream past through stainless space,

J2 was a value of light stream past through the investigated cell chemical substance [13]. Values J1 and J2 were showed by amperemetre.

Relative index of total substance's quantity Q in cell nucleus and cytoplasm was defined:

where D was an optical density, S was the cell's area [14].

Cell and nucleus dimensions were measured by ocular micrometer in relative units.

Statistic processing

Mean arithmetic value M was defined:

where M was row's separate members, n was number of row’s members[15].

Mean value of quadratic derivation G was defined:

where Mmax and Mmin are the largest and the least values, Mmax – Mmin is an amplitude, K was the coefficient corresponded to an amplitude defined in Ermolaev’s table [16 ].

Mistake of mean value m was calculated:

The mean value of mistake of compared mean values was determined:
where mc was the mean value of control group, me was the mean value of experimental group.

The index of essential difference T between two groups was defined:

where Mc is the mean arithmetic of control group, Me is the mean arithmetic of experimental group.

Differences P were found in Student’s Table.