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Mechlorethamine (NTG) effects onthe erythrocytic and leukocytic blood parametersduring experimentally induced pleuritis in rats

Ireneusz Ca³kosiñski1, Joanna Rosiñczuk-Tonderys1, Katarzyna Dzierzba2,Justyna Bazan2, Monika Ca³kosiñska3, Jacek Majda4,Maciej Dobrzyñski5, Agnieszka Bronowicka-Szyde³ko2

1Department of Nervous System Diseases, The Faculty of Health Science, Wroclaw Medical University, Bartla 5,PL 51-618 Wroc³aw, Poland

2Department of Medical Biochemistry, Wroclaw Medical University, Cha³ubiñskiego 10, PL 50-368 Wroc³aw,Poland

3Outpatient Clinic Medcom in Wojkowice, Wojkowice 28B, PL 55-020 ¯urawina, Poland

4Department of Diagnostics Laboratory, 4thMilitary Academic Hospital in Wroclaw, Weigla 5, PL 53-114 Wroc³aw,Poland

5Department of Conservative Dentistry and Pedodontics, Wroclaw Medical University, Krakowska 26,PL 50-425 Wroc³aw, Poland

Correspondence: Ireneusz Ca³kosiñski, e-mail: [email protected]

Abstract:

Background: According to cytotoxic and mutagenic properties, nitrogranulogen (NTG) changes the character of inflammatory re-actions. Our previous studies have shown that NTG can enhance immunological defense reactions, because of its high affinity toDNA, and causes disorders in the synthesis of acute phase proteins (e.g., haptoglobin, transferrin, fibrinogen and complement pro-tein C3) [15]. The aim of the current studies was to determine the influence of three different NTG doses: 5 µg/kg b.w. (body weight),50 µg/kg b.w. and 600 µg/kg b.w. (cytotoxic dose) on the values of hematological blood parameters: RBC, HGB, HCT, RDW, MCV,MCH, MCHC, PLT, MPV, PCT, PDW, WBC, NEUT, LYMPH, MONO, EOS and BASO in pleuritis-induced rats.Methods: The animals were randomized into five groups: Group I – control group; Group II – IP (induced pleuritis) group; Group III– NTG5 group; Group IV – NTG50 group; Group V – NTG600 group. The blood was collected from all the groups at the 24th h,48th h, and 72nd h after the initiation of the carrageenin-induced inflammatory reaction.Results: These investigations have revealed that NTG administered at the dose of 5 µg/kg b.w. caused the drop of the leukocyte andlymphocyte numbers and the rise of the neutrophil number at the 72nd h of the experimental-induced inflammatory reaction. Moreo-ver, the dose of: 5 µg/kg b.w. was an immunomodulatory property and it also increased the erythrocytic parameters. On the contrary,NTG applied at the doses of 50 µg/kg and 600 µg/kg b.w. contributed to the drop of both: the erythrocytic and leukocytic parametersduring the whole time of the inflammatory reaction.Conclusions: The results suggest that nitrogranulogen affects the erythropoiesis.

Key words:

mechlorethamine, nitrogranulogen, NTG, inflammatory reaction, hematological parameters

650 Pharmacological Reports, 2012, 64, 650�672

Pharmacological Reports2012, 64, 650�672ISSN 1734-1140

Copyright © 2012by Institute of PharmacologyPolish Academy of Sciences

Abbreviations: BASO – basophiles, b.w. – body weight, EOS– eosinophiles, HCT – hematocrit, HGB – hemoglobin, LYMPH– lymphocytes, MCH – mean corpuscular hemoglobin, MCHC– mean corpuscular hemoglobin concentration, MCV – meancorpuscular volume, MONO – monocytes, MPV – mean plate-let volume, NEUT – neutrophils, NTG – nitrogranulogen(mechlorethamine), PCT – thrombocrit, PDW – platelet distri-bution width, PLT – platelets, RBC – erythrocytes, RDW – redblood cell distribution width, WBC – leukocytes

Introduction

Mechlorethamine (N-methyl-2,2’-dichlorodiethylamine;Nitrogranulogen; Chlormethine; NTG) is a derivativeof the nitrogen mustard. This alkylating (therapeutic)agent is used in the treatment of cancer. NTG is espe-cially effective in the treatment of the cutaneousT-cell lymphoma (CTCL), e.g., Hodgkin’s disease,multiple myeloma and brain tumors [7–9, 19, 23, 25,27, 35, 43, 47]. Mechlorethamine, chlorambucid mel-phalan, cyclophosphamide and ifosfamide are someof the clinically important nitrogen mustards used inpresent therapies [8, 25, 35, 45, 48]. However, theyare highly toxic and cause several side-effects, affect-ing more or less all tissues and some secondary skincancer e.g., basal and squamous cell carcinoma [7, 21,45, 53]. The cytotoxic and anticancer activities ofNTG are mainly associated with their ability to forminter-strand crosslinks in DNA molecules [48]. TheNTG is not selective toward neoplastic cells and inter-acts rapidly with growing cells producing the undesir-able effects, e.g., bone marrow depression [7, 21, 45].This drug is applied in the combination with the che-motherapy called MOPP together with vincristine(oncovin), procarbazine and prednisone for the treat-ment of Hodgkin’s disease and other lymphomas [9,11, 25, 41, 45].

According to many studies, NTG can be an effectivedrug in many diseases treatment e.g., connective tissuediseases (CTD) such as: chronic, multisystemic, con-nective tissue pathologies, e.g., scleroderma, systemiclupus erythematosus, Sjögren’s syndrome, mixed con-nective tissue diseases and rheumatoid arthritis [8, 16,34]. NTG is also used in treatment of autoimmunediseases characterized by the fibrosis of the skin andvisceral organs, the impairment of blood vessels andthe auto-antibodies production [8, 16, 18, 34]. Thecommon syndromes in patients with connective tissuediseases are collagen and elastin damages caused by

the chronic inflammation [1–6, 10, 24, 28, 33, 37, 44].NTG administrated at the dose of 10–50 µg/kg b.w.has anti-inflammatory properties contrary to signifi-cantly higher doses which are cytotoxic [1–6, 56].Application of these chemotherapeutical agents causethe general inflammatory reactions e.g., fever, pain,swelling and significantly influence the intensifica-tion of repair processes e.g., wound healing by secondintention [1–6].

Small doses of NTG have been applied into multi-ple sclerosis (MS) treatment and caused the intensi-fied synthesis and the endogenous glucocorticoster-oids release [24, 49]. NTG can be also used in the an-tibiotic therapies [4]. The intensified phagocytosisand the rise of the neutrophiles number is observedafter the administration of NTG at the dose of1–10 µg/kg b.w. into guinea pigs and mice [20, 24].This humoral response has been monitored afterSRBC, ovalbumin immunizations and LPS applica-tion [20, 24, 32, 38]. The same reaction is observedafter NTG administration at the dose of 100 µg/kgb.w. [30, 52]. According to the previous studies, theenhancement of the humoral response against erythro-cyte antigen (SRBC) occurs between the 72nd and96th h of the pleuritis, after five-fold NTG applicationat the dose of 5 µg/kg b.w. [14]. Moreover, these stud-ies have pointed to the rise of the hemolysins from theIgG class (antibody producing cells, PFC) and anti-SRBC antibodies [14, 38, 39]. These studies have alsoshown the decreases of PFC and anti-SRBC valuesafter NTG administration at the dose of 600 µg/kgb.w. [14, 39, 54]. The similar immunosuppressivemode of action is characteristic after using NTG at thedose of 250–500 µg/kg b.w. [32, 53–55]. Further-more, NTG influences on ATP-ase activity in lym-phocytes membranes [42]. The radical drop of theneutrophiles number and swelling are characteristicsyndroms after NTG application into guinea pigswithout earlier zymosan administration what is usedfor induction of the inflammatory reaction [56].

NTG shows alkylating properties associated withinhibition of DNA synthesis and causes the inhibitionof mitotic divisions in somatic cells, what is displayedby rapid growth. Therefore, the effects of NTG actioncan be perceived as the negative effect of the hemato-poiesis processes. Clinical effects of alkylating drugs– derivatives of mustard gas, associated with myelo-suppressive properties, have been well documentedfor years. According to research of Rancewicz [43],the dose of NTG of 10 µg/kg b.w. administered once

Pharmacological Reports, 2012, 64, 650�672 651

Effects of different NTG doses on hematological blood parameters in induced pleuritis in ratsIreneusz Ca³kosiñski et al.

per day during 7 days contributes to reduction of thenumber of NK cells, reduction of the number of leu-kocytes, but does not change the number of erythro-cytes and platelets. Other studies performed on rab-bits and mice have shown that NTG used at the doseof 1–10 µg/kg causes the increase of phagocytosis andthe number of neutrophils [20]. Many of clinical ob-servations point to the fact that drugs based on mustardgas can cause an acute bone marrow. The bone marrowdamage causes changes in the amount of granulocytes,which reaches its lowest level on 6thday of the NTG ap-plication, while the decrease of blood platelets occursin 4th week after the NTG application [17].

The previous studies of NTG influence on bio-chemical blood parameters in rats with experimental-induced pleuritis have shown the necessity of execut-ing the analysis of hematological blood parameters inrats with the inflammation [15]. The current studiesshow the dynamics of hematological blood parame-ters after NTG administration at three different dosesof: 5, 50 and 600 µg/kg b.w. during 72nd h of experi-mental-induced pleuritis.

Materials and Methods

Experimental animals

The studies were performed on female rats from theBuffalo inbreeding strain (aged 8–10 weeks, 120–140 gb.w.). Because some diagnostic parameters are de-pendent on individual features such as age, sex orstrain (under invariable environmental factors), onlyBuffalo females (with similar mass and age) wereused in these experiments [15]. These animals wererelated and have the same sex in order to show thesame reactivity on the inflammatory factor and to ob-tain results with similar standard deviation. The ratswere bred in the Department of Pathomorphology inWroclaw Medical University and kept under the sameconditions: they were in polystyrene cages with metallids (6 animals to each cage). The experiments werecarried out in air-conditioned rooms – the temperatureoscillated between 21 and 22°C, the humidity of airwas 62–63% [15]. The rats were fed using the stan-


Fig. 1. The diagram of the pleuritis induction in rats with temporal monitoring of the hematological number during inflammatory reaction afterNTG administration at three different doses [15]

dard diet „Murigran”, and they received water ad libi-

tum. The experiments with animals were permitted byThe Local Bioethics Council for Animal Experiments(permission number: 23/2001).

The animals were divided into five groups what isshown in the Figure 1 [15].1. CONTROL – control group of 24 female rats with-out inflammation. This is the physiological group with-out carrageenan and NTG applications. The blood wascollected at the 72nd h of the experiment;2. IP group – group of 60 female rats with inducedpleuritis caused by a single intrapleural injection of0.15 ml of 1% carrageenan solution (Sigma-Aldrich),given at the 1st min of the experiment;3. NTG5 group – group of 60 female rats injectedintravenously (iv) with NTG (Polfa Warszawa SA) ata dose of 5 µg/kg b.w. with a 1%-solution carrageenanat the 2nd, 24th and 48th h after the pleuritis induction.The blood was collected after a single NTG applica-tion (at the 24th h), after a double NTG application (atthe 24th and 48th h) and after a threefold NTG appli-cation (at the 24th, 48th and 72nd h) after the inflam-mation initiation;4. NTG50 group – group of 60 female rats injected iv

with NTG (Polfa Warszawa SA) at a dose of 50 µg/kgb.w. at the 2nd, 24th and 48th h after the pleuritis induc-tion using a 1% solution of carrageenan. The bloodwas collected after a single NTG application (at the24th h) after a double NTG application (at the 24th and48th h) and after a threefold NTG application (at the24th, 48th and 72nd h) after the inflammation initiation;5. NTG600 group – group of 60 female rats injectediv with NTG at a dose of 600 µg/kg b.w. at the 2nd,24th and 48th h after the pleuritis induction using a 1%carrageenan solution. The blood was collected aftera single NTG application (at the 24th h), after a doubleNTG application (at the 24th and 48th h) and aftera threefold NTG application (at the 24th, 48th h and72nd h) after the inflammation initiation.

Materials

Carrageenan (Sigma) extracted from Chondruschris-

pus algae was dissolved before the experiments in0.9% NaCl solution (Polfa, Warszawa SA). Next, thissolution was injected into the 4–5 intercostal space onthe right site.

NTG (Polfa,Poland) was dissolved in water (aquapro injectione, Polpharma, Poland) and injected intothe tail vein.

The blood for the studies was collected from all thegroups of experimental animals at the 24th, 48th and72nd h after the induction of the experimental pleuritis.The biological material was collected from rats underpentobarbital-induced anesthesia (Biochemie GmbH,Germany), which was administered by intraperitonealinjection at a dose of 30 mg/kg b.w. Next, the abdomi-nal cavity was opened and catheters and needles of2 mm diameters were inserted into aorta for collectingblood into standardized polypropylene test tubes(Sarstedt Ltd., USA).

Methods

The process of the inflammation in rats treated withcarrageenan and proper NTG dose was monitored byestimation of the following hematological parameters:erythrocytes (RBC), hemoglobin (HGB), hematocrit(HCT), red-blood cell distribution width (RDW), meancorpuscular volume (MCV), mean corpuscular hemo-globin (MCH), mean corpuscular hemoglobin con-centration (MCHC), platelets (PLT), mean plateletvolume (MPV), thrombocrit (PCT), platelet distribu-tion width (PDW), leukocytes (WBC), neutrophilsSysmex XT 1800i (NEUT), lymphocytes (LYMPH),monocytes (MONO), eosinophiles (EOS), basophiles(BASO). The basic hematological parameters weremarked using standard diagnostics tests and SysmexXT 1800i hematological analyzer (Sysmex PolandLtd.) at the Diagnostic Laboratory of the 4th MilitaryAcademic Hospital in Wroc³aw, Poland.

Statistical analysis

The hematological parameter values in rat blood wereanalyzed using Statistica 7.0 (StatSoft Ltd.). Thearithmetic means of parameters (X) for determinednumber of animals taking part in experiment (N),standard deviation (D), minimal (MIN) and maximum(MAX) value ranges of parameters were computed.After checking whether the data conformed to a nor-mal distribution (comparing the variable histogramswith a Gaussian distribution chart), particular groupswere compared using Student’s t-test with takingBonferroni correction under consideration to deter-mine levels of significance (p – statistical significancetowards IP group).



Methods of statistical correlation evaluation

In order to evaluate statistical correlation betweengroups treated by different NTG doses at 24th, 48th

and 72nd h of pleuritis, the analysis of variance(ANOVA) was carried out. The ANOVA test allowedfor effective evaluation of the influence of NTG doseson various hematological parameters in different ex-perimental time intervals. Before conducting theANOVA test, normal distribution was verified byShapiro-Wilk’s test on the significance level a = 0.05.Significance of differences between two subgroupswas verified by Tukey HSD for unequal N test andLSD test. The Tukey assay was also used to distinguishgroups that demonstrate changeability. Similarly to thevariation assay, the variation mean is found equal if thep-value of the assay is lower than the steady probabil-ity of a type I error. In another case, we observed a sta-tistically significant difference.

Results

Analysis of obtained erythrocytic parameters

in experimentally induced pleuritis in rats

after NTG administration at doses 5, 50 and

600 µg/kg b.w.

Erythrocytes

The RBC level for the IP group relative to the controlgroup is lower during the total experimental time (ex-cept the first blood measurement at the 24th h inwhich RBC level for IP group is higher than in controlgroup) and the RBC value for the IP group is stilldropping within 72 h of the experiment (Fig. 2a). Thedecrease in the RBC level compared to the controland IP groups is observed in the NTG5, NTG50, andNTG600 groups at the 24th h after pleuritis initiationin the following order: NTG5 < NTG50 < NTG600 (Tab.1). The second NTG administration at the doses of 50and 600 µg/kg b.w. causes a drastic drop in the RBCconcentrations between the 24th and 48th h relative tothe control and IP groups and these differences arestatistically significant (Tab. 2). On the other hand,the second NTG administration at the dose of 5 µg/kgb.w. increases the RBC level between the 24th and48th h relative to the IP group and this difference isalso statistically significant (Tab. 2). The third injec-

tion of NTG into the NTG50 and NTG600 groupcauses further RBC drop (NTG50 and NTG600groups vs. both: control and IP groups) between the48th and 72nd h of the inflammation (Fig. 2a). Thesedifferences are statistically significant (Tab. 2). Simi-larly, the third injection of NTG into the NTG5 groupcauses a continuous increase of the RBC concentra-tion between the 48th and 72nd h (NTG5 group vs.

both: control and IP groups) and the RBC levelreaches higher value in NTG5 group than in the con-trol group at the 72nd h of the experiment (Tab. 1).The differences of the RBC concentration betweenNTG5 and the control groups as well as betweenNTG5 and the IP groups are statistically significant atthe 72nd h of the inflammation (Tab. 2). The decreasein the RBC level is observed in the NTG50 group inevery blood analysis compared to the control and IPgroups. Two-way ANOVA (the mean vs. time andNTG dose) shows an essential difference in the varia-tion of NTG doses. Tukey’s tests demonstrate the sta-tistically essential differences in the control groupcompared to the IP, NTG50 and NTG600 groups.A negative correlation between the RBC number inthe monitored duration of pleuritis in relation to thedose used in this group: NTG 600 µg/kg b.w. (Fig. 2b,Tab. 3) is indicated. Moreover, correlation betweenthe RBC number and the size of NTG dose is nega-tive.

These data probably point to pro-inflammatoryproperties of NTG at a dose of 50 µg/kg b.w. (Fig.2a).The statistically significant rise in the concentra-tion of RBC in relation to the IP group at the 48th and


Fig. 2a. The influence of three NTG doses on the RBC number duringexperimentally induced pleuritis in rats



Tab. 1. The mean values [X] and standard deviation [D] of selected hematological parameters between individual groups of rats withcarrageenin-induced inflammation after triple NTG application at three different doses of: 5, 50 and 600 µg/kg b.w.

Hematologicalparameter

Control group IP group NTG5 group NTG50 group NTG600 group

X D X D X D X D X D

RBC(´ 106/µl)

8.52 0.26 24 h: 8.6948 h: 7.6872 h: 7.51

0.220.210.54

24 h: 8.5548 h: 8.0872 h: 9.07

1.020.700.27

24 h: 6.7248 h: 5.7572 h: 5.50

0.831.082

11

24 h: 5.9948 h: 5.8872 h: 4.50

0.400.831.41

HGB(g/dl)

15.74 0.57 24 h: 15.7848 h: 14.172 h: 13.60

0.500.420.86

24 h: 15.4348 h: 14.4272 h: 16.60

1.891.040.42

24 h: 12.2848 h: 10.2372 h: 9.54

1.391.843.69

24 h: 11.1348 h: 10.7072 h: 8.13

0.731.502.80

HCT(%)

43.02 1.22 24 h: 43.4848 h: 39.0572 h: 38.18

1.081.422.70

24 h: 42.0348 h: 40.7272 h: 46.08

5.042.681.20

24 h: 36.3648 h: 32.1672 h: 29.55

2.273.828.60

24 h: 36.0748 h: 33.6872 h: 27.53

1.754.018.74

RDW(%)

13.39 0.96 24 h: 13.0848 h: 13.0872 h: 13.4

0.380.280.43

24 h: 12.6348 h: 13.2272 h: 12.90

0.230.320.30

24 h: 15.3648 h: 16.6172 h: 15.89

2.722.603.70

24 h: 15.4548 h: 15.4572 h: 14.90

1.781.532.69

MCV(fl)

50.49 0.82 24 h: 49.8548 h: 50.8872 h: 51.05

0.800.851.11

24 h: 48.8848 h: 50.4272 h: 50.92

1.341.451.14

24 h: 54.5848 h: 56.7372 h: 55.70

5.385.336.43

24 h: 60.6248 h: 57.4572 h: 61.15

1.241.582.18

MCH(pg)

18.11 0.32 24 h: 18.1348 h: 18.2772 h: 18.18

0.390.340.45

24 h: 17.8548 h: 17.9372 h: 18.22

0.450.400.22

24 h: 18.3148 h: 17.8572 h: 17.34

0.940.570.44

24 h: 18.7048 h: 18.2872 h: 17.94

0.320.220.67

MCHC(g/dl)

35.91 0.66 24 h: 36.3848 h: 35.8872 h: 35.60

0.370.290.30

24 h: 36.3348 h: 35.6372 h: 35,87

0.160.500.62

24 h: 33.7448 h: 31.6672 h: 31.48

2.722.603.70

24 h: 30.8548 h: 31.7572 h: 29.35

0.580.870.78

PLT(´ 103/µl)

789.36 86.2 24 h: 642.3348 h: 712.5072 h: 600.33

10994.2153

24 h: 794.0048 h: 697.3372 h: 854.00

29.410366.8

24 h: 535.7848 h: 673.8872 h: 636.75

117228193

24 h: 425.1748 h: 643.5072 h: 624.38

181110239

MPV(fl)

6.29 0.27 24 h: 6.7348 h: 6.7772 h: 7.20

0.260.120.85

24 h: 6.4248 h: 6.3772 h: 6.30

0.130.380.00

24 h: 6.7148 h: 6.7472 h: 6.93

0.780.390.47

24 h: 6.1048 h: 6.1872 h: 6.16

0.200.410.54

PCT(%)

0.51 0.06 24 h: 0.4348 h: 0.4872 h: 0.43

0.070.060.09

24 h: 0.5148 h: 0.4672 h: 0.55

0.020.080.04

24 h: 0.3748 h: 0.4672 h: 0.45

0.080.150.14

24 h: 0.2848 h: 0.4372 h: 0.40

0.120.060.15

PDW(%)

54.93 1.91 24 h: 52.0548 h: 51.3572 h: 51.35

2.540.651.02

24 h: 53.3648 h: 54.8072 h: 54.87

0.601.930.69

24 h: 55.3448 h: 56.7972 h: 53.40

6.252.704.53

24 h: 60.7848 h: 59.4072 h: 61.45

1.561.885.87

WBC(´ 103/µL)

5.22 1.33 24 h: 7.7348 h: 7.7572 h: 8.74

0.671.932.33

24 h: 5.4048 h: 6.8672 h: 5.69

0.531.341.51

24 h: 9.1948 h: 5.8372 h: 7.39

8.123.553.53

24 h: 6.9448 h: 3.4672 h: 1.99

1.590.870.85

NEUT(%)

25.21 6.28 24 h: 47.0248 h: 24.5572 h: 24.12

17.33.286.68

24 h: 42.3248 h: 22.7772 h: 53.22

14.216.114.0

24 h: 42.4348 h: 38.8372 h: 32.65

18.416.414.0

24 h: 61.1048 h: 38.5872 h: 42.02

7.0412.722.6

LYMPH(%)

52.36 5.81 24 h: 29.6248 h: 41.8772 h: 48.45

5.915.0910.5

24 h: 32.5248 h: 51.9572 h: 21.43

6.8913.32.74

24 h: 35.5748 h: 39.8972 h: 46.09

8.1410.29.67

24 h: 29.4248 h: 48.1072 h: 44.72

7.2910.823.8

MONO(%)

12.94 3.61 24 h: 14.6248 h: 20.5072 h: 16.18

3.403.979.47

24 h: 16.5348 h: 15.3872 h: 14.92

10.94.598.41

24 h: 13.1048 h: 13.2972 h: 12.99

10.98.958.99

24 h: 4.8048 h: 5.2272 h: 6.75

1.790.601.91

EOS(%)

2.33 1.99 24 h: 2.2748 h: 2.1472 h: 2.82

1.382.142.27

24 h: 2.8048 h: 2.7272 h: 3.52

1.261.912.30

24 h: 4.6148 h: 1.5172 h: 2.15

4.991.342.47

24 h: 0.9548 h: 0.5572 h: 0.40

0.630.140.09

BASO(%)

0.41 0.14 24 h: 0.3048 h: 0.3072 h: 0.43

0.060.090.14

24 h: 0.2848 h: 0.3572 h: 0.42

0.180.140.08

24 h: 0.3348 h: 0.1672 h: 0.21

0.300.080.10

24 h: 0.3348 h: 0.2772 h: 0.58

0.080.050.31


Tab. 2. Statistical data analysis performed by using Student’s t-test with Bonferroni correction to determine levels of significance (the indica-tions: * p < 0.05, ** p < 0.01 and *** p < 0.001. NS – not significant.)

Compared groups using Student’s t-test with Bonferroni correction

Hematological parameter Control groupvs. IP group

IP groupvs. NTG5 group



RBC (´ 106/µl) 24 h: NS48 h: ***72 h: ***

24 h: NS48 h: NS72 h: ***

24 h: ***48 h: **72 h: *

24 h: ***48 h: ***72 h: ***

HGB (g/dl) 24 h: NS48 h: ***72 h: ***

24 h: NS48 h: NS72 h: ***

24 h: ***48 h: ***72 h: *

24 h: ***48 h: ***72 h: **

HCT (%) 24 h: NS48 h: ***72 h: ***

24 h: NS48 h: NS72 h: ***

24 h: ***48 h: **72 h: *

24 h: ***48 h: *72 h: *

MCV (fl) 24 h: NS48 h: NS72 h: NS

24 h: NS48 h: NS72 h: NS

24 h: NS48 h: *72 h: NS

24 h: ***48 h: ***72 h: ***

RDW (%) 24 h: NS48 h: NS72 h: NS

24 h: *48 h: NS72 h: *

24 h: *48 h: ***72 h: **

24 h: *48 h: **72 h: NS

MCH (pg) 24 h: NS48 h: NS72 h: NS

24 h: NS48 h: NS72 h: NS

24 h: NS48 h: NS72 h: **

24 h: *48 h: NS72 h: NS

MCHC (g/dl) 24 h: NS48 h: NS72 h: NS

24 h: NS48 h: NS72 h: NS

24 h: *48 h: **72 h: *

24 h: ***48 h: ***72 h: ***

PLT (´ 103/µl) 24 h: **48 h: NS72 h: **

24 h: **48 h: NS72 h: **

24 h: NS48 h: NS72 h: NS

24 h: *48 h: NS72 h: NS

MPV (fl) 24 h: **48 h: ***72 h: **

24 h: *48 h: *72 h: *

24 h: NS48 h: NS72 h: NS

24 h: ***48 h: **72 h: *

PCT (%) 24 h: *48 h: NS72 h: *

24 h: *48 h: NS72 h: *

24 h: NS48 h: NS72 h: NS

24 h: *48 h: NS72 h: NS

PDW (%) 24 h: *48 h: ***72 h: ***

24 h: NS48 h: **72 h: ***

24 h: NS48 h: ***72 h: NS

24 h: ***48 h: ***72 h: **

WBC (´ 103/µl) 24 h: ***48 h: **72 h: ***

24 h: ***48 h: NS72 h: *

24 h: NS48 h: NS72 h: NS

24 h: NS48 h: ***72 h: ***

NEUT (%) 24 h: ***48 h: NS72 h: NS

24 h: NS48 h: NS72 h: **

24 h: NS48 h: NS72 h: NS

24 h: NS48 h: *72 h: NS

LYMPH (%) 24 h: ***48 h: **72 h: NS

24 h: NS48 h: NS72 h: ***

24 h: NS48 h: NS72 h: NS

24 h: NS48 h: NS72 h: NS

MONO (%) 24 h: NS48 h: ***72 h: NS

24 h: NS48 h: NS72 h: NS

24 h: NS48 h: NS72 h: NS

24 h: NS48 h: ***72 h:*

EOS (%) 24 h: NS48 h: NS72 h: NS

24 h: NS48 h: NS72 h: NS

24 h: NS48 h: *72 h: NS

24 h: NS48 h: **72 h: *

BASO (%) 24 h: NS48 h: NS72 h: NS

24 h: NS48 h: NS72 h: NS

24 h: NS48 h: *72 h: *

24 h: NS48 h: NS72 h: NS

72nd h of pleuritis is noticed only for the NTG5 group(Tab. 1) and these data suggest therapeutic propertiesof this NTG dose and its anti-inflammatory proper-ties. The drastic drop of the RBC level in the NTG600group during the whole time of the experiment pointsto strong pro-inflammatory properties of NTG at thedose of 600 µg/kg b.w.

Hemoglobin

A significant drop in the HGB level in the IP grouprelative to the control group is observed between the24th and 72nd h of the experiment (Fig. 2c). Pleuritisinitiation causes insignificant rise of the HGB concen-tration in IP group during the first 24 h (IP group vs.

control group). Each NTG dose causes a drop in thelevels of HGB relative to the IP and control groups at

the 24th h of pleuritis initiation (Tab. 1). This decreaseoccurs in the following order: NTG5 < NTG50 <NTG600 and differences between the HGB level inNTG50 and NTG600 relative to both IP and controlgroup are statistically significant during the first 24th

h of pleuritis (Tab. 2). The second NTG application atthe 48th h causes a subsequent HGB drop in theNTG50 and NTG600 groups compared to both: IPand control group and these differences are statisti-cally significant (Tab. 2). However, a contrary effectis observed in the NTG5 group at the 48th h – theHGB concentration rises in this group relatively to IPgroup, the HGB level is still significantly lower inNTG5 group than in the control group (Fig. 2c). Thethird NTG application is responsible for a further dropin the levels of HGB in the NTG50 and NTG600groups, and the HGB significant rise in the NTG5



Fig. 2c. The influence of three NTG doses on the HGB (g/dl) duringexperimentally induced pleuritis in rats

Tab. 3. Correlation coefficients r between erythrocyte parameters and inflammation duration, from the 24th to 72nd h after the induction of theexperimental pleuritis. Statistically significant dependences are bolded. Abbreviations: RBC – erythrocytes, HGB – hemoglobin, HCT – hema-tocrit, MCV – mean corpuscular volume, MCH – mean corpuscular hemoglobin, NTG5, NTG50, NTG600 – group of rats injected iv with nitro-granulogen at a dose of 5, 50 and 600 µg/kg b.w., respectively

Group RBC HGB HCT MCV MCH

NTG5 r = 0.273 r = 0.326 r = 0.434 r = 0.563 r = 0.402

p = 0.417 p = 0.188 p = 0.072 p = 0.015 p = 0.098

NTG50 r = –0.373 r = –0.462 r = –0.502 r = 0.100 r = –0.510

p = 0.051 p = 0.013 p = 0.006 p = 0.614 p = 0.006

NTG600 r = –0.544 r = –0.563 r = –0.555 r = 0.093 r = –0.618

p = 0.020 p = 0.015 p = 0.017 p = 0.715 p = 0.006

Fig. 2b. Correlation diagram and regression model and linear corre-lation coefficient RBC 600 with duration of experimental pleuritis; r –correlation coefficient; p – statistical significance

group at the 72nd h of the inflammatory reaction (Fig.2c). Two-way ANOVA (the mean vs. time and NTGdose) shows an essential difference in the variation ofNTG doses. Tukey’s tests demonstrate the statisticallyessential differences in the control group compared tothe IP, NTG50 and NTG600 groups and in the IPgroup compared to the NTG50 and NTG600 groups.It is indicated that hemoglobin (HGB) concentrationreveals a negative correlation during the monitoringprocess of pleuritis in the groups administered withthe NTG doses: 50 and 600 µg/kg b.w. (Tab. 3).

A single injection of 5 µg NTG/kg b.w. does notcause a significant change in the inflammation char-acter. Just two- and three-time doses of NTG5 en-hance anti-inflammatory properties of NTG at thedose of 5 µg NTG/kg b.w. and increase HGB level.The significant lower HGB level in NTG50 groupcompared to IP group during the whole time of the ex-periment suggests pro-inflammatory properties ofNTG at the dose of 50 µg NTG/kg b.w. A rapid HGBdecrease observed in the NTG600 group compared toIP group between the 24th and 72nd h of pleuritis pointto strong anti-inflammatory properties of this NTGdose (Fig. 2c).

Hematocrit

A statistically significant HCT drop in the IP grouprelative to the control group is observed only at the72nd h of the experiment (Tab. 2). The first NTG ap-plication causes the HCT drop in all the NTG groupsin relation to the IP group at the 24th h of pleuritis.This decrease occurs in the following order: NTG5 <NTG50 < NTG600 (Fig. 2d, Tab. 1). The second NTGadministration causes a contrary effect in NTG5group: HCT rises in this group compared to IP groupat the 48th h but this change is not statistically signifi-cant (Tab. 2). On the other hand, the second NTG ad-ministration into NTG50 and NTG600 groups is re-sponsible for further HCT drop. Differences betweenNTG50 or NTG600 groups and both: control and IPgroups are statistically significant at the 48th h ofpleuritis (Tab. 2) and these decreases occur in the fol-lowing order: NTG50 < NTG600 (Fig. 2d). The thirdNTG administration enhances properties of each NTGdose: HCT consistently rises in NTG5 group andreaches higher value than HCT in control and IPgroups. These differences are statistically significant(Tab. 2). Moreover, the third NTG administrationcauses a continuous HCT drop in both NTG50 and

NTG600 groups compared to the IP and controlgroups at the 72nd h of pleuritis (Fig. 2d, Tab. 1).These differences are also statistically significant(Tab. 2). Two-way ANOVA (the mean vs. time andNTG dose) shows an essential difference in the varia-tion of NTG doses. Tukey’s tests demonstrate the sta-tistically essential differences in the control groupcompared to the IP, NTG50 and NTG600 groups andin the IP group compared to the NTG50 and NTG600groups. Furthermore, a negative correlation of hema-tocrit value in the monitored duration of pleuritis inrelation to the used NTG doses: 50 and 600 µg/kgb.w. (Tab. 3) is indicated.

The statistically significant rise of HCT in NTG5group relative to both: IP and control groups is no-ticed only at the 72nd h of pleuritis (Tab. 2) and this re-sult suggests anti-inflammatory properties of NTG atthe dose of 5 µg/kg b.w. The decrease of the HCT inthe NTG50 and NTG600 groups in relation to both IPand control groups during the whole time of the ex-periment point to pro-inflammatory properties ofNTG at the dose of 50 µg/kg or 600 µg/kg b.w.

Red blood cell distribution width

A drop in the RDW value in the IP group relative tothe control group is observed during the whole time ofthe experiment (Fig. 2e, Tab. 1) but these differencesare not statistically significant. NTG administration atthe dose of 5 µg/kg b.w. does not cause significantchanges of RDW for NTG5 group compared to both:control and IP groups in the whole time of pleuritis.On the other hand, NTG administration at the dose of


Fig. 2d. The influence of three NTG doses on the HCT (%) during ex-perimentally induced pleuritis in rats

50 or 600 µg/kg b.w. is responsible for RDW rises inNTG50 and NTG600 groups in relation to IP and con-trol groups at the 24th h of pleuritis. These differencesare statistically significant (Tab. 2). The second NTGapplication at the 48th h causes a subsequent RDWrise in the NTG50 and NTG600 groups compared toboth: IP and control group and these differences arestatistically significant (Tabs. 1, 2). Although a simi-lar effect is observed in the NTG5 group at the 48th h– the RDW value rises in this group relatively to bothIP and control groups, the differences are statisticallyinsignificant (Tab. 2). The third NTG applicationcauses the RDW drop in all NTG groups between the48th and 72nd h of the inflammation (Fig. 2e). The dif-ference in the RDW values are statistically significant

between NTG50 or NTG600 groups and both controland IP groups at the 72nd h of pleuritis (Tab. 2).Lower values of RDW in NTG5 group than in IPgroup could point to pro-inflammatory properties ofNTG at the dose of 5 µg/kg b.w. However, differencesin RDW values between NTG5 and IP group are sta-tistically insignificant and administration of NTG atthe dose of 5 µg/kg b.w. does not change RDW valuesin the inflammatory reaction. On the other hand, NTGadministered at the doses of: 50 or 600 µg/kg b.w. sig-nificantly increases RDW values and, in consequence,shows anti-inflammatory properties of these NTGdoses at the 24th and 72nd h of the pleuritis comparedto IP group.

Significantly higher RDW value at the 48th h inNTG50 group suggests strong anti-inflammatoryproperties of NTG at the dose of 5 µg NTG/kg b.w.(Fig. 2e, Tab. 1, Tab. 2).

Mean corpuscular volume

Pleuritis initiation causes MCV drop during the first24 h in IP group compared to the control group butthis change is not statistically significant (Fig. 2f,Tab. 1, Tab. 2). The MCV value rises in IP group rela-tively to the control group between the 24th and 72nd

h of pleuritis but these differences are not statisticallysignificant (Tab. 2). Pleuritis initiation does not sig-nificantly influence the MCV value (the IP group vs.

the control group). The first NTG application causesthe MCV drop in the NTG5 group compared to both:IP and control groups at the 24th h of the inflamma-tion. A contrary effect is observed in NTG50 andNTG600 groups in which the first NTG application atthe dose of 50 or 600 µg/kg b.w., respectively, causesstatistically significant increase of MCV value in rela-tion to the IP and control group at the 24th h of pleuri-tis (Fig. 2f, Tab. 2). Although the second NTG ad-ministration causes the increase of MCV value inNTG5 and NTG50 groups at the 48th h, the differenceis statistically significant only between NTG50 andboth IP and control groups (Tab. 2). A contrary effectis observed in NTG600 group – the second NTG ad-ministration is responsible for the drop of MCV valueat the 48th h in this group. However, the MCV valuefor NTG600 group at the 48th h is still significantlyhigher than in both IP and control group. The thirdNTG administration at the dose of 5 µg/kg b.w. insig-nificantly increases the MCV value at the 72nd h inNTG5 group in relation to the control and IP groups



Fig. 2f. The influence of three NTG doses on the MCV (fl) during ex-perimentally induced pleuritis in rats

Fig. 2e. The influence of three NTG doses on the RDW (%) during ex-perimentally induced pleuritis in rats

(Tab. 2). On the other hand, the third NTG administra-tion into the NTG600 group is responsible for the sig-nificant increase of the MCV value at the 72nd h ofpleuritis and its value for the NTG600 group is muchhigher than in other groups at the same time (Fig. 2f).NTG administration at the dose of 5 µg/kg b.w. doesnot change significantly MCV values during thewhole time of the experiment. The rise of the NTGdose up to 50 µg/kg b.w. causes the increase of theMCV values in the NTG50 group compared to both:control and IP groups during the whole time of the in-flammation. Two-way ANOVA (the mean vs. timeand NTG dose) does not show an essential differencein the variation of NTG doses. Tukey’s tests demon-strate the statistically essential differences in theNTG600 group compared only to the IP. Furthermore,it is indicated that erythrocyte volume MCV duringexperimental pleuritis reveals a positive correlationtowards the duration with the used dose – 5 µg/kgb.w. (Tab. 3). Moreover, NTG administration at thedose of 600 µg/kg b.w. is responsible for the consider-able increase of the MCV values in the NTG600group at three measurement points what suggestsstrong anti-inflammatory properties of this NTG dose.

Mean corpuscular hemoglobin

The MCH level for the IP group relative to the controlgroup is insignificantly higher during the total experi-mental time, so pleuritis initiation does not change theMCH value (Fig. 2g, Tab. 1). The increase of theMCH level compared to the control and IP groups isobserved in all NTG groups at the 24th h after pleuri-tis initiation in the following order: NTG5 < NTG50< NTG600. However, a statistically significant differ-ence at the 24th h of pleuritis is observed only forNTG600 group compared to the IP or control groups.Although the second NTG administration at the dosesof 50 and 600 µg/kg b.w. causes a drop of the MCHlevel between the 24th and 48th h relative to the con-trol and IP groups, these differences are not statisti-cally significant (Tab. 2). Moreover, the second NTGadministration at the dose of 5 µg/kg b.w. does not in-fluence significantly the MCH level between the 24th

and 48th h of pleuritis (Tab. 2). The third NTG injec-tion into NTG50 and NTG600 groups causes furtherMCH drop (NTG50 and NTG600 groups vs. both:control and IP groups) between the 48th and 72nd h ofthe inflammation (Fig. 2g, Tab. 1). These differencesare statistically significant only for the NTG50 group

(Tab. 2). Similarly, the third injection of NTG into theNTG5 group causes a continuous increase of theMCH level between the 48th and 72nd h (NTG5 groupvs. both: control and IP groups) but this change is notstatistically significant (Tab. 2). The differences of theMCH level between NTG5 and the control groups aswell as between NTG5 and the IP groups are not sta-tistically significant in the whole time of the inflam-mation (Tab. 2). The lack of NTG influence on MCHvalues is also observed for NTG administered at dosesof 50 and 600 µg/kg b.w – the difference in the MCHvalue between NTG50 and both: control and IP groupis statistically significant only at the 72nd h of pleuri-tis (Tab. 2). Two-way ANOVA (the mean vs. time andNTG dose) does not show an essential difference inthe variation of NTG doses. Tukey’s tests demonstratethe statistically essential differences in the NTG50group compared only to the IP group at 72nd h ofpleuritis. Moreover, it is indicated that there occursa negative correlation MCH in relation to duration ofinflammation among the groups administered withNTG doses: 50 and 600 µg/kg b.w. (Tab. 3).

Mean corpuscular hemoglobin concentration

The MCHC level for the IP group relative to the con-trol group is insignificantly lower during the total ex-perimental time (except the first blood measurementat the 24th h in which MCHC level for IP group ishigher than in the control group) and the MCHC valuefor the IP group is still dropping within 72 h of the ex-periment (Fig. 2h, Tab. 1). The decrease of the MCHClevel compared to the control and IP groups is ob-


Fig. 2g. The influence of three NTG doses on the MCH (pg) duringexperimentally induced pleuritis in rats

served in all NTG groups at the 24th h after pleuritisinitiation in the following order: NTG5 < NTG50 <NTG600. However, a statistically significant differ-ence at the 24th h of pleuritis is observed only forNTG50 and NTG600 group compared to the IP orcontrol groups (Tab. 2). The second NTG administra-tion at the doses of 50 µg/kg b.w. causes a drop in theMCHC level between the 24th and 48th h relative tothe control and IP groups and this difference is statis-tically significant (Tab. 2). Although the second NTGadministration at the dose of 600 µg/kg b.w. causesa rise of the MCHC level, a value of this parameterfor NTG600 group is still much lower than for thecontrol and IP groups at the 48th h of pleuritis. More-over, the second NTG administration at the dose of5 µg/kg b.w. decreases the MCHC level between the24th and 48th h relative to the IP group but this differ-ence is not statistically significant (Tab. 2). The thirdinjection of NTG into the NTG50 and NTG600 groupcauses further MCHC drop (NTG50 and NTG600groups vs. both: control and IP groups) between the48th and 72nd h of the inflammation (Fig. 2h). Thesedifferences are statistically significant (Tab. 2). On thecontrary, the third NTG injection into the NTG5group causes a continuous increase of the MCHClevel between the 48th and 72nd h (NTG5 group vs.

both: control and IP groups) but this difference is notstatistically significant (Tabs. 1, 2). The differences ofthe MCHC level between NTG5 and the controlgroups as well as between NTG5 and the IP groupsare not statistically significant in the whole time of theinflammation (Tab. 2). The decrease in the MCHClevel is observed in the NTG50 group in every blood

analysis compared to the control and IP groups. Two-way ANOVA (the mean vs. time and NTG dose) doesnot show an essential difference in the variation ofNTG doses. Tukey’s tests demonstrate the statisticallyessential differences in the NTG50 and NTG600group compared to the IP.

These data probably point to pro-inflammatoryproperties of NTG at the dose of 50 µg/kg b.w. (Fig.2h). The most drastic decrease in the MCHC level ob-served in the NTG600 group compared to anothergroups in the whole time of pleuritis suggests strongpro-inflammatory properties of NTG at the dose of600 µg/kg b.w. (Fig. 2h).

Platelets

A statistically significant difference of PLT level inthe IP group compared to the control group is ob-served in the whole time of the experiment (Tab. 2).Although PLT value rises at the 48th h in IP group, itis still significantly lower than in the control group(Fig. 3a, Tab. 1). The first NTG application at thedose of 5 µg/kg b.w. causes a significant increase ofthe PLT concentration in the NTG5 group comparedto IP group and PLT level achieves a similar value asin the control group at the 24th h of pleuritis. On thecontrary, the first NTG application at the dose of 50 or600 µg/kg b.w. causes the significant PLT drop inNTG50 and NTG600 groups in relation to the IPgroup at the 24th h of pleuritis. This decrease occursin the following order: NTG50 < NTG600 (Fig. 3a,Tab. 1). The second NTG administration causesa contrary effect: PLT level rises in NTG50 andNTG600 groups and decreases in NTG5 group com-pared to the changes occurred at the 24th h of pleurits(Tab. 1). However, the PLT level in all NTG groups isstill significantly lower than in IP group at the 48th hof the experiment (Fig. 3a, Tab. 2). Differences inPLT level between NTG50 or NTG600 groups andboth: control and IP groups are statistically significantat the 48th h of pleuritis (Tab. 2) and these decreasesoccur in the following order: NTG50 < NTG600 (Fig.3a, Tab. 1). The third NTG administration enhancesproperties of each NTG dose: PLT consistently risesin NTG5 group and reaches higher value than PLT incontrol and IP groups. These differences are statisti-cally significant (Tab. 2). Moreover, the third NTGadministration causes a continuous PLT drop in bothNTG50 and NTG600 groups compared to the IP andcontrol groups at the 72nd h of pleuritis (Fig. 3a).



Fig. 2h. The influence of three NTG doses on the MCHC (g/dl) duringexperimentally induced pleuritis in rats

These differences are also statistically significant(Tab. 2).

The statistically significant rise of PLT in NTG5group compared to the IP group is noticed at the 24th

and 72nd h of pleuritis (Tab. 2) and these results sug-gest anti-inflammatory properties of NTG at the doseof 5 µg/kg b.w. The significant lower PLT level in theNTG50 and NTG600 groups in relation to both IP andcontrol groups during the whole time of the experi-ment point to pro-inflammatory properties of NTG atthe dose of 50 or 600 µg/kg b.w.

Mean platelet volume

The MPV value for the IP group relative to the controlgroup is higher during the total experimental time andthese differences are statistically significant at eachblood measure point of the experiment (Fig. 3b, Tab. 1).The decrease of the MPV value for NTG groups com-pared to the IP group is observed at the 24th h afterpleuritis initiation in the following order: NTG50 <NTG5 < NTG600. The second NTG administration atthe doses of 5 µg/kg b.w. causes further MPV drop inNTG5 group and this value is lower than in IP groupat the 48th h of pleuritis. On the contrary, the secondNTG administration at the dose of 50 and 600 µg/kgb.w. causes a continuous insignificant increase ofMPV level in NTG50 and NTG600 groups comparedto changes occurred at the 24th h of pleurits (Tab. 1).The third NTG administration enhances properties ofeach NTG dose: MPV consistently rises in NTG50group and reaches the highest MPV value comparedto MPV values of other groups at the 72nd h of pleuritis.

These differences are statistically significant (Tab. 2).The third injection of NTG into the NTG5 andNTG600 groups causes further MPV drop (NTG5 andNTG600 groups vs. IP group) between the 48th and72nd h of the inflammation (Fig. 3b). These differ-ences are statistically significant (Tab. 2). Moreover,the third NTG injection at the dose of 5 µg/kg b.w.causes that the MPV value in the NTG5 group is al-most the same as in the control group.

These data probably point to anti-inflammatoryproperties of NTG at the dose of 5 µg/kg b.w. Thelower MPV value observed in the NTG600 group inevery blood analysis than in the IP and control groupssuggests that NTG at the dose of 600 µg/kg b.w. hasstronger anti-inflammatory properties than NTG at thedose of 5 µg/kg b.w. (Fig. 3b). On the other hand,three-fold NTG administration at the dose of 50 µg/kgb.w. does not change the character of the inflammation.

Thrombocrit

The PCT value for the IP group relative to the controlgroup is lower during the total experimental time andthese differences are statistically significant at eachblood measure point of the experiment, except for thesecond blood measurement at the 48th h of pleuritis(Fig. 3c, Tabs. 1, 2). The statistically significant lowerPCT values for NTG50 and NTG600 groups com-pared to the IP and control groups are observed at the24th h after pleuritis initiation in the following order:NTG50 < NTG600 (Tabs. 1, 2). On the other hand,the first NTG administration at the dose of 5 µg/kgb.w. increases PCT value in NTG5 group compared to


Fig. 3a. The influence of three NTG doses on the PLT number duringexperimentally induced pleuritis in rats

Fig. 3b. The influence of three NTG doses on the MPV (fl) during ex-perimentally induced pleuritis in rats

IP group and this value is the same as in the controlgroup at the 24th h (Fig. 3c). The contrary effect ofNTG doses occurs after the second NTG administra-tion: at the dose of 5 µg/kg b.w. it causes PCT de-crease in NTG5 group and this value is insignificantlylower than in the control and IP groups at the 48th h ofpleuritis. Moreover, the second NTG administration atthe doses of 50 and 600 µg/kg b.w. is responsible forthe increase of PCT value for NTG50 and NTG600groups compared to changes occurred at the 24th h ofpleurits (Tab. 1). However, these values are still lowerthan in the IP and control groups at the 48th h of theexperiment (Tab. 2). These differences are not statisti-cally significant. The third NTG administrationcauses the drop of PCT values in NTG50 andNTG600 groups and these values are still signifi-cantly lower than in the control group at the 72nd h ofthe experiment. On the other hand, the third NTG ad-ministration at the dose of 5µg/kg b.w. causes furtherincrease of PCT value in NTG5 group compared tochanges occurred in this group at the 48th h of pleu-ritis (Fig. 3c, Tab. 1). Moreover, the PCT value inNTG5 group is higher than in both: control and IPgroups at the 72nd h of the experiment.

Higher values of PCT in NTG5 group than in the IPgroup and similar as in the control group could pointto anti-inflammatory properties of NTG at the dose of5 µg/kg b.w. Furthermore, approximate PCT values inNTG50 group and in the IP group in each blood meas-urement suggest that NTG at the dose of 50 µg/kgb.w. does not change the character of the inflamma-tion. The significant lower PCT value in the NTG600

group at the 24th h than in the IP group point to pro-inflammatory properties of NTG at the dose of 600 µg/kg b.w. (Fig. 3c).

Platelet distribution width

Tthe PDW value for the IP group relative to the con-trol group is lower during the total experimental timeand these differences are statistically significant ateach blood measure point of the experiment (Tabs. 1, 2).The PDW value for the IP group is almost on the samelevel during the whole time of pleuritis (Fig. 3d). Theincrease of the PDW value compared to the IP groupis observed in all NTG groups at the 24th h after pleu-ritis initiation in the following order: NTG5 < NTG50< NTG600 (Fig. 3d). However, NTG application atthe doses of 5 and 50 µg/kg b.w. causes an insignifi-cant increase of the PDW value in NTG5 and NTG50groups, respectively, compared to IP group and PDWvalues in these groups and achieve a similar value asin the control group at the 24th h of pleuritis. Moreo-ver, the first NTG application at the dose of 600 µg/kgb.w. causes the significant PDW rise in NTG600groups in relation to both: control and IP groups at the24th h of pleuritis (Tabs. 1, 2). The second NTG ad-ministration is responsible for further PDW rises inNTG5 and NTG50 groups compared to the changesoccurred at the 24th h of pleurits in these groups(Tab. 1). On the other hand, the second NTG admini-stration into NTG600 group does not significantlychange PDW value at the 48th h in this group com-



Fig. 3c. The influence of three NTG doses on the PCT% during ex-perimentally induced pleuritis in rats

Fig. 3d. The influence of three NTG doses on the PDW% during ex-perimentally induced pleuritis in rats

pared to the changes occurred at the 24th h of pleuritis(Fig. 3d). The third NTG application causes insignifi-cant increases of PDW value in NTG5 and NTG600groups at the 72nd h of the inflammation compared tothe IP group. Furthermore, the third NTG applicationat the dose of 50 µg/kg b.w. is responsible for thePDW drop in the NTG50 group at the 72nd h and thePDW value achieves a similar value as in the controlgroup at the 72nd h of the experiment (Tab. 1).

The statistically significant rise of PDW in NTG5and NTG50 groups compared to the IP group noticedduring the whole time of pleuritis and similar PDWvalues in these groups as in the control group suggestanti-inflammatory properties of NTG at doses of 5and 50 µg/kg b.w. The significant higher PDW valuesin the NTG600 group in relation to both IP and con-trol groups during the whole time of the experimentpoint to pro-inflammatory properties of NTG at the of600 µg/kg b.w.

The analysis of obtained leukocytic parameters

in experimentally induced pleuritis in rats

after NTG administration at doses 5, 50 and

600 µg/kg b.w.

Leukocytes

The WBC number for the IP group in relation to thecontrol group is higher during the total experimentaltime and these differences are statistically significantat each blood measure point of the experiment (Tabs.1, 2). Furthermore, WBC number for the IP group isalmost on the same level at the 24th h and 48th h andincreases at the 72nd h of pleuritis (Fig. 4a). The de-crease of the WBC number in NTG5 and NTG600groups compared to the IP group is observed at the24th h after pleuritis initiation (Fig. 4a, Tab. 1). How-ever, the WBC value in the NTG5 group is similar asin the control group and much higher in NTG600group compared to the control group at the 24th h ofexperimental time. Moreover, NTG application at thedose of 50 µg/kg b.w. significantly increases theWBC number in NTG50 group compared to both con-trol and IP groups at the 24th h of the inflammation(Tab. 1). The second NTG administration causes con-trary effects: WBC number at the 48th h significantlyincreases in the NTG5 group and drastically drops inthe NTG50 and NTG600 groups compared to thechanges occurred at the 24th h of pleurits in thesegroups (Fig. 4a). The WBC number in NTG50 and

NTG600 groups is statistically significantly lowerthan WBC number in IP group at the 48th h of pleurits(Tab. 2). However, WBC number in NTG600 group issignificantly lower than in the control group and onsimilar level in NTG50 and the control group. Thethird NTG application causes insignificant drop ofWBC number in NTG5 group compared to thechanges occurred at the 48th h of pleurits and thisWBC value is similar to WBC number in the controlgroup and considerably lower than WBC numberin the IP group at the 72nd h of experimental time(Tab. 1). Moreover, the third NTG application ata dose 50 µg/kg b.w. is responsible for the WBC in-crease in NTG50 group compared to the changes oc-curred at the 48th h of the inflammation and this WBCvalue is similar as in the WBC number of IP group atthe 72nd h. On the other hand, the third NTG applica-tion at a dose of 600 µg/kg b.w. causes significantdrop of WBC number in NTG600 group and thisvalue is the lowest compared to WBC number ofother groups at the 72nd h of experimental time. Two-way ANOVA (the mean vs. time and NTG dose)shows an essential difference in the variation of NTGdoses. Tukey’s tests demonstrate the statistically es-sential differences in the control group compared tothe IP, NTG5 and NTG600 groups and in the IP groupcompared to the NTG5 and NTG600 groups. Further-more, in the NTG600 group a negative correlation be-tween the WBC number and duration of experimentalpleuritis is indicated (Fig. 4b, Tab. 4).


Fig. 4a. The influence of three NTG doses on the WBC number duringexperimentally induced pleuritis in rats

The statistically significant lower WBC number inNTG5 group noticed during the whole time of pleuri-tis and similar WBC values in this group as in thecontrol group suggest anti-inflammatory properties ofNTG at doses of 5 µg/kg b.w. Furthermore, the ap-proximate WBC number in NTG50 group and in theIP group in each blood measurement suggest thatNTG at the dose of 50 µg/kg b.w. does not change thecharacter of the inflammation. The significant lowerWBC number in the NTG600 group in relation toboth IP and control groups at the 48th and 72nd h of theexperiment points to pro-inflammatory properties ofNTG at the dose of 600 µg/kg b.w.

Neutrophils

The NEUT number for the IP group in relation to thecontrol group is significantly higher at the 24th h andbalances on the same level between the 48th h and72nd h of the experimental time (Tab. 1). The firstNTG application causes the NEUT number increasein all NTG groups in the following order: NTG5 £

NTG50 < NTG600 (Fig. 4c). The NEUT number ishigher in all NTG groups compared to the controlgroup and significantly higher in NTG600 group inrelation to the IP group at the 24th h of the experiment.NEUT number in the second blood measurement issignificantly lower in all groups with induced inflam-mation (NTG groups and IP group) compared toNEUT number of these groups at the 24th h of pleuri-tis. The NEUT value in NTG5 group at the 48th h ofthe experiment is similar as in both: control and IPgroups. On the other hand, after the second NTG ad-ministration at doses of 50 and 600 µg/kg b.w. theNEUT number in NTG50 and NTG600 groups is sta-tistically significantly higher than in both: control and



Fig. 4b. Correlation diagram and regression model and linear corre-lation coefficient WBC 600 with duration of experimental pleuritis;r – correlation coefficient; p – statistical significance

Fig. 4c. The influence of three NTG doses on the NEUT number dur-ing experimentally induced pleuritis in rats

Tab. 4. Correlation coefficients r between leukocyte parameters and inflammation time, from the 24th to 72nd h after the induction of the experi-mental pleuritis. Statistically significant dependences are bolded. Abbreviations: WBC – leukocytes, NEUT – neutrophils, LYMPH – lympho-cytes, MONO – monocytes, EOS – eosinophiles, NTG5, NTG50, NTG600 – group of rats injected iv with nitrogranulogen at a dose of 5, 50 and600 µg/kg b.w., respectively

Group WBC NEUT LYMPH MONO EOS

NTG5 r = 0.093 r = 0.241 r = –0.305 r = –0.086 r = 0.146

p = 0.713 p = 0.335 p = 0.219 p = 0.735 p = 0.564

NTG50 r = –0.149 r = –0.240 r = 0.567 r = –0.209 r = –0.149

p = 0.450 p = 0.248 p = 0.003 p = 0.317 p = 0.476

NTG600 r = –0.873 r = –0.450 r = 0.426 r = 0.487 r = –0.549

p < 0.001 p = 0.061 p = 0.078 p = 0.040 p = 0.018

IP groups (Tab. 2). The third NTG application causesstatistically significant rise of NEUT number inNTG5 group compared to NEUT number after thesecond blood measurement at the 48th h of pleuritis.This value is also statistically significant in relation tothe NEUT value in the IP and control group at the72nd h of the inflammation (Tab. 1). Moreover, thethird NTG application at the dose of 50 µg/kg b.w. isresponsible for the NEUT number decrease in NTG50group compared to NEUT number at the 48th h of theexperiment. However, this value is still higher thanNEUT number in both: control and IP group at the72nd h of pleuritis. A contrary effect is observed inNTG600 group at the 72nd h – NEUT number in thisgroup increases and it is still statistically significantlyhigher than in IP and control groups. Two-wayANOVA (the mean vs. time and NTG dose) does notshow an essential difference in the variation of NTGdoses. Tukey’s tests demonstrate the statistically es-sential differences only in the NTG5 group comparedto the IP at 72nd h of pleuritis. In the NTG50 group nocorrelation between the number of NEUT and themonitoring duration of pleuritis is indicated (Tab. 4).Lower values of NEUT number in NTG5 group thanin NEUT number in IP group at the 24th and 48th h ofthe experiment point to anti-inflammatory propertiesof NTG at the dose of 5 µg/kg b.w.

Lymphocytes

The LYMPH number for the IP group in relation to thecontrol group is lower during the total experimental timeand its value increases between the 24th h and 72nd h ofthe experiment (Fig. 4d, Tab. 1). Furthermore, LYMPHnumber for the IP group is almost on the same level asin the control group at the 72nd h of pleuritis (Fig. 4d).The first NTG administration at doses of 5 and 50 µg/kgb.w. causes LYMPH number increase in NTG5 andNTG50 groups compared to LYMPH number in the IPgroup at the 24th h of the pleuritis. However, theseLYMPH values in NTG5 and NTG50 groups are stillstatistically significantly lower than LYMPH value inthe control group at the 24th h of the experiment(Tab. 2). Moreover, the first NTG administration at thedose of 600 µg/kg b.w. does not change LYMPHnumber during pleuritis and LYMPH value in NTG600group is almost the same as LYMPH value in the IPgroup at the 24th h of the inflammation (Fig. 4d).LYMPH number in the second blood measurement issignificantly higher in all groups with induced inflam-

mation (NTG groups and IP group) compared toLYMPH number of these groups at the 24th h of pleuri-tis. The LYMPH values in NTG5 and NTG600 groupsat the 48th h of the experiment are similar as in the con-trol group and statistically significantly higher than inthe IP group (Tabs. 1, 2). Furthermore, the second NTGadministration at the dose of 50 µg/kg b.w. causes thatLYMPH number in NTG50 and IP groups is almost onthe same level. The third NTG application causes statis-tically significant decrease of LYMPH number in NTG5group compared to the changes occurred at the 48th h ofpleuritis. The LYMPH value of the NTG5 group is alsosignificantly lower than LYMPH number in both: con-trol and IP groups at the 72nd h of pleuritis (Tabs. 1, 2).Moreover, the third NTG application at a dose 50 µg/kgb.w. is responsible for the LYMPH number increase inNTG50 group compared to the changes occurred at the48th h of the inflammation and this LYMPH value issimilar as in the LYMPH number of both: control and IPgroups at the 72nd h. On the other hand, the third NTGapplication at a dose 600 µg/kg b.w. causes insignificantdrop of LYMPH number in NTG600 group compared tothe changes occurred at the 48th h of the inflammationand this LYMPH value is similar as in the LYMPHnumber of both: control and IP groups at the 72nd h.Two-way ANOVA (the mean vs. time and NTG dose)does not show an essential difference in the variation ofNTG doses. Tukey’s tests demonstrate the statisticallyessential differences only in the NTG5 group comparedto the IP at 72nd h of pleuritis. Moreover, in theNTG50 group a positive correlation between thenumber of LYMPH and the monitoring duration ofpleuritis is indicated (Tab. 4).


Fig. 4d. The influence of three NTG doses on the LYMPH numberduring experimentally induced pleuritis in rats

Monocytes

The MONO value for the IP group in relation to thecontrol group is higher during the total experimentaltime and these differences are statistically significantat each blood measure point of the experiment (Tab.1, Tab. 2). Moreover, the MONO value for the IPgroup increases between the 24th and 48th h and de-creases between 48th and 72nd h of pleuritis (Fig. 4e).The MONO number in the NTG5 group increases atthe 24th h after pleuritis initiation compared to thecontrol and IP groups. The first NTG application atthe doses of 50 µg/kg b.w. causes an insignificantdrop of MONO numbers in the NTG50 group com-pared to the IP group at the 24th h of the inflammationand this value is similar as MONO number in the con-trol group. On the other hand, the first NTG applica-tion at the doses of 600 µg/kg b.w. causes the statisti-cally significant decrease of MONO numbers inNTG600 group in relation to both: control and IPgroups (Tabs. 1, 2). The MONO number in the NTG5group insignificantly decreases at the 48th h of pleuri-tis compared to the changes in this group occurred atthe 24th h. Although, the MONO level in NTG5 groupis statistically significantly lower than in the IP groupat the 48th of the inflammation, its value in the NTG5group is still significantly higher than in the controlgroup (Tab. 2). Moreover, the second NTG admini-stration does not change significantly MONO numberin NTG50 and NTG600 groups at the 48th h comparedto the changes occurred in these groups at the 24th hof pleuritis (Fig. 4e). The third NTG application

causes insignificant decreases of MONO number inNTG5 and NTG50 groups at the 72nd h of the inflam-mation compared to the changes in these groups oc-curred at the 48th h of pleuritis. Furthermore, MONOvalues in NTG5 and NTG50 groups are lower thanMONO value in IP group at the 72nd h of the inflam-mation. However, the third NTG application only atthe dose of 50 µg/kg b.w. causes that MONO numberin NTG50 achieves a similar value as in the controlgroup at the 72nd h of the experiment (Tab. 1). More-over, the third NTG application at the dose of 600 µg/kg b.w. is responsible for further increase of MONOnumber in NTG600 group compared to the changes oc-curred in this group at the 48th h of pleuritis. However,the MONO level in NTG600 group is still statisticallysignificantly lower than in the control and IP group atthe 72nd h of pleuritis (Tabs. 1, 2). Two-way ANOVA(the mean vs. time and NTG dose) does not showan essential difference in the variation of NTG doses.Tukey’s tests demonstrate the statistically essential dif-ferences in the IP compared to NTG600 group. Moreo-ver, in this group a positive correlation occurred in theMONO number in relation to the monitoring durationof the inflammation (Tab. 4).

The statistically significant drop of MONO numberin NTG5 and NTG50 groups compared to the IPgroup noticed during the whole time of pleuritis (ex-cept the MONO value in NTG5 group after first NTGapplication) and similar MONO values in thesegroups as in the control group suggest anti-inflam-matory properties of NTG at doses of 5 and 50 µg/kgb.w. The drastic lowering of MONO number in theNTG600 group in relation to both IP and controlgroups during the whole time of the experiment pointto strong anti-inflammatory properties of NTG at theof 600 µg/kg b.w.

Eosinophiles

The EOS number in the IP group is almost the sameas in the control group during all experimental time(Fig. 4f). The EOS number in the NTG5 groupslightly increases at the 24th h after pleuritis initiationcompared to the control and IP groups. The first NTGapplication at the doses of 50 µg/kg b.w. causes thestatistically significant rise of EOS numbers in theNTG50 group compared to the control and IP groupsat the 24th h of the inflammation. On the other hand,the first NTG application at the doses of 600 µg/kgb.w. causes a statistically significant decrease of EOS



Fig. 4e. The influence of three NTG doses on the MONO number dur-ing experimentally induced pleuritis in rats

numbers in NTG600 group in relation to both: controland IP groups (Tabs. 1, 2). The EOS number in theNTG5 group insignificantly decreases at the 48th h ofpleuritis compared to the changes in this group oc-curred at the 24th h. Moreover, the second NTG ad-ministration changes significantly EOS number inNTG50 and NTG600 groups at the 48th h compared tothe changes occurred in these groups at the 24th h ofpleuritis (Fig. 4f). Both of these values are statisticallysignificantly lower in NTG50 and NTG600 groupsthan in the control and IP groups at the 48th h of theexperiment. The third NTG application causes signifi-cant increase of EOS number in NTG5 and NTG50groups at the 72nd h of the inflammation compared tothe changes in these groups occurred at the 48th h ofpleuritis. Furthermore, EOS value in NTG5 group isstatistically significantly higher than EOS value in thecontrol and IP group at the 72nd h of the inflamma-tion. However, the third NTG application only at thedose of 50 µg/kg b.w. causes that EOS number inNTG50 achieves a similar value as in the controlgroup at the 72nd h of the experiment (Tab. 1). More-over, the third NTG application at the dose of 600µg/kg b.w. is responsible for further decrease of EOSnumber in NTG600 group compared to the changesoccurred in this group at the 48th h of pleuritis. How-ever, the EOS level in NTG600 group is still statisti-cally significantly lower than in the control and IPgroups at the 72nd h of pleuritis (Tab. 2). Two-wayANOVA (the mean vs. time and NTG dose) does notshow an essential difference in the variation of NTGdoses. Tukey’s tests demonstrate the statistically es-

sential differences only in the NTG600 group com-pared to the IP group. In this group a negativecorrelation in the EOS number occurred in relation tothe monitoring duration of the inflammation (Tab. 4).

The similar EOS values in the NTG5 and controlgroups during the whole time of the experiment (ex-cept the EOS number in NTG5 group after the thirdNTG application) suggest anti-inflammatory proper-ties of NTG at the dose of 5 µg/kg. The drastic lower-ing of EOS number in the NTG600 group in relationto both IP and control groups during the whole time ofthe experiment points to strong pro-inflammatoryproperties of NTG at the of 600 µg/kg b.w.

Basophiles

The BASO number in the IP group is lower than inthe control group during all experimental time, exceptthe third blood measurement in which these values arealmost on the same level (Fig. 4g). The first NTG ap-plication into all NTG groups does not significantlychange BASO number in these groups compared toBASO number in the IP group at the 24th h of pleuritis(Tabs. 1, 2). Moreover, the first NTG application intoall NTG groups causes that BASO value in thesegroups is significantly lower than in the controlgroup. The BASO number in the NTG5 group insig-nificantly increases at the 48th h of pleuritis comparedto the changes in this group occurred at the 24th h.Moreover, the second NTG administration signifi-cantly decreases BASO number in NTG50 group atthe 48thh compared to the changes occurred in thesegroups at the 24th h of pleuritis (Fig. 4g). BASOnumber in NTG50 group at the 48th h of the inflam-


Fig. 4g. The influence of three NTG doses on the BASO number dur-ing experimentally induced pleuritis in rats

Fig. 4f. The influence of three NTG doses on the EOS number duringexperimentally induced pleuritis in rats

mation is the lowest in relation to BASO number inother groups. The second NTG application in the doseof 600 µg/kg b.w. does not significantly changeBASO value in the NTG600 group compared to thechanges occurred in this group at the 24th h of pleu-ritis (Fig. 4g). The third NTG application causes sig-nificant increase of BASO number in all NTG groupsat the 72nd h of the inflammation compared to thechanges in these group occurred at the 48th h of pleu-ritis. Furthermore, BASO value in NTG5 group is al-most the same as in the control and IP groups at the72nd h of the inflammation. Although the third NTGapplication at the dose of 50 µg/kg b.w. causes the in-crease of BASO number in NTG50, BASO value inthis group is statistically significantly lower thanBASO number in the control group at the 72nd h ofthe experiment (Tabs. 1, 2). Moreover, the third NTGapplication at the dose of 600 µg/kg b.w. is responsi-ble for the drastic increase of BASO number inNTG600 group compared to the changes occurred inthis group at the 48th h of pleuritis.

The BASO level in NTG600 group is significantlyhigher than in the control and IP groups at the 72nd hof pleuritis (Tab. 1).

The similar BASO values in the NTG5 and controlgroups during the whole time of the experiment (exceptthe EOS number in NTG5 group after the first NTG ap-plication) suggest anti-inflammatory properties of NTGat the dose of 5 µg/kg. The significantly lower BASOnumber in the NTG50 group in relation to both IP andcontrol groups during the whole time of the experiment(except the EOS number in NTG5 group after the firstNTG application) point to strong pro-inflammatoryproperties of NTG at the of 50 µg/ kg b.w.

Discussion

The NTG application in three different doses maychange the course of the experimentally induced pleu-ritis which dynamics is determined by the hemato-logical and biochemical parameters in the blood se-rum [13, 15]. According to many studies, the NTGadministration into rats in the dose of 1–10 µg/kg b.w.shows immunostimulative properties [26, 27, 39, 40,51, 54]. The aim of the current studies was to check ifsmall NTG doses could contribute in modulatory wayto change the inflammation reaction course. Thechanges in the amount and proportion of leukocytes in

blood and in concentration of proteins of the acutephase point to the turning point of the inflammationdynamics which occurs between the 24th and 72nd hof this process, what is proved by changes of bio-chemical parameters such as AspAT, AlAT, urea, cre-atinine, C3 and C4 proteins [15].

In the current studies we have observed changes inbehavior of some inflammation blood parameters afterNTG administration in three different doses: 5, 50 and600 µg/kg b.w. The amount of the third NTG dose wasused according to several scientific communicationswhere NTG in cytotoxic doses (400–600 µg/kg b.w.) in-fluenced some hematological and biochemical parame-ters [15, 22, 31]. In the current studies, as controlgroups, animals with induced pleuritis by carragenan(the IP group) were used as well as intact animals (thecontrol group). NTG has a high hepatogenic affinity andinteracts with DNA during cell divisions [12]. TheseNTG properties cause that NTG can significantly influ-ence the dynamics of the inflammatory reaction deter-mined by the hematological parameters changes.

The estimation of hematological parameter

changes during the inflammatory reaction

course after the NTG administration at the

doses of: 5, 50 and 600 µg/kg b.w.

Experiments on sheep carried out by Magid showedthat the five-day NTG administration at the dose of8000 mg/kg resulted in bone marrow hypoplasia [36].The current studies using 600 µg NTG/kg b.w. asa maximum NTG dose, during three-days of pleuritis,have indicated changes in hematological blood pa-rameters and have not shown the hypoplastic changesin the bone marrow, although parameters such as RBCand HTC showed significant differences compared toRBC and HTC values in animal groups treated bylower NTG doses: 5 µg/kg or 50 µg/kg b.w.

The threefold application of NTG at the dose of5 µg/kg b.w. caused rises of the RBC number, HTCvalue and HGB concentration at the 72nd h of the in-flammatory reaction without changes of other eryth-rocytic parameters. These characteristic changes oc-curred in NTG groups in relation to IP group can beexplained by the induction of the erythropoietic pro-cesses. The NTG application at the dose of 5 µg/kgb.w. caused also the drop of the WBC number in theperipheral blood in NTG5 group compared to IPgroup and achieves WBC number as in the controlgroup. These data probably point to anti-inflammatoryproperties of NTG at the dose of 5 µg/kg b.w. Moreover,



the percentage content of neutrophiles increased, con-trary to the percentage content of lymphocytes whichdecreased. The PLT number in NTG5 group signifi-cantly rises compared to the PLT value in the IP group(expect the second blood measurement in which PLTlevel in IP group was insignificantly higher than in theNTG5 group). Probably, NTG at the dose of 5 µg/kgb.w. has anti-inflammatory properties.

The NTG application at the doses of 50 and600 µg/kg b.w. caused the inverted effects than NTGapplication at the dose of 5 µg/kg b.w. The decreasesof the RBC number, HTC values and HGB concentra-tion noticed between the 24th and 72nd h of theexperimentally-induced pleuritis in rats pointed topro-inflammatory properties of NTG of these doses.In these groups, the MCV and RDW parameters in-creased and MCHC parameter decreased. Probably,10-fold and 120-fold doses of NTG influence signifi-cantly the erythropoiesis during the inflammation.However, only a few scientific communications haveshown the immune-simulative NTG influence onerythrocytic parameter changes [22, 51, 52]. A smalldose of NTG injected into NTG5 group decreased thepro-inflammatory rise of the WBC number. Further-more, the NTG application at the dose of 600 µg/kgb.w. significantly decreased the leukocytic reactionduring induced pleuritis. According to other studiescarried out on guinea pig and rats, NTG have causedthe weakness of neutrophiles reaction in the tissuedamage site [50]. The current studies have shown thatthreefold NTG administration at the dose of 50 µg/kgb.w. probably caused the neutrophilia at the 72nd h ofthe inflammatory reaction. The significant drop of theWBC number between 48th h and 72nd h of the in-duced pleuritis in NTG600 group is a result of cyto-toxic action of such an enormous NTG dose [29].Moreover, cytotoxic action of NTG at the dose of600 µg/kg b.w. causes intensified WBC loss and defec-tive their regeneration. NTG at the dose of 600 µg/kgb.w. is also responsible for the decrease of the percent-age content of eosinophiles and monocytes.

Moreover, correlation in the NTG600 group in re-lation to the number of WBC and eosinophilias ischaracterized by a negative value, which supports animmunosuppressive dose NTG600 used during theexperimental pleuritis. Application of NTG in the ex-perimental pleuritis at the dose of 50 µg/kg b.w. tem-porarily changes the character of WBC response tosuperiority of lymphocytes.

Conclusions

The current studies have indicated the decrease of theerythrocyte number, hematocrit value and hemoglo-bin concentration caused by experimentally-inducedpleuritis in rats. The hemolysis of erythrocytes in theinflammatory nidus and disseminated intravascularcoagulation (DIC) are the main reasons for the in-flammation [46]. Probably, the erythropoietic processdoes not undergo the disturbances completely what ispointed by a lack of changes of MCV, MCH andMCHC parameters.

The proportions of leukocytic forms in the periph-eral blood changed significantly in the course of theinflammatory reaction [57]. The WBC drop is con-cerned with the leukocytic form accumulation in theinflammation nidus.

The erythrocytic parameters in the NTG5 group in-creased at the 72nd h hour of the induced pleuritis. Theleukocytosis was not observed. The leukocytic pictureinversed at the 72nd h of the inflammatory reaction: theneutrophiles number increased opposite to lympho-cytes which number significantly dropped. The signifi-cant rise of the platelets was also noticed at that time.

The NTG application at the dose of 50 µg/kg b.w.caused the decrease of the RBC, HCT and HGB pa-rameters in earlier phases of the inflammatory reac-tion, similarly to NTG600 group of experimental ani-mals. Moreover, the loss of erythrocytes took place inNTG50 and NTG600 groups. The rise of MCV, MCHand MCHC values in both: NTG50 and NTG 600groups probably suggests that NTG influence erythro-poiesis.

Supporting information:

The publication is based on habilitation thesis “The influenceof tocopherol on diagnostic indexes of inflammatory reaction in ratsundergoing dioxin exposition” by Ireneusz Ca³kosiñski.

Acknowledgment:

The project was financially supported by grants No. PB-768 andPB-327 from Wroclaw Medical University.

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Received: July 17, 2011; in the revised form: December 29, 2011;accepted: January 12, 2012.


mechlorethamine (ntg) effects on the erythrocytic and ...if-pan.krakow.pl/pjp/pdf/2012/3_650.pdf ·...

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