TIME COURSE OF MICE LUNG MECHANICAL AND HISTOLOGICAL DAMAGE BY MICROCYSTIN-LR

Cagido VR, Soares RM, Fonseca DB, Azevedo SMFO, Faffe DS, Zin WA
Laboratory of Respiration Physiology Laboratory of Ecophysiology and Toxicology of Cyanobacteria Carlos Chagas Filho Biophysics Institute Federal University of Rio de Janeiro Brazil

Introduction
Cyanobacterias are photosynthetic prokaryotic microorganisms with biochemical and cellular organizations similar to bacterias. Public health concern regarding cyanobacteria centers on the ability of many species to produce cyanotoxins. Wherever conditions of temperature, light and nutrient status are conducive, surface waters may host increased growth of cyanobacteria. When such proliferation is dominated by a single or a few species, the phenomenon is referred as cyanobacterial bloom.

Introduction

Baltic Sea, Stockholm, Sweden.

Jacarepagu Lake, Rio de Janeiro, Brazil Crawley Bay, Swan-Canning Estuary, Australia. O Globo 13/08/01

Introduction
Microcystins (MC) are the most commonly found cyanotoxins released into the water by cyanobacteria. These toxins are cyclic heptapeptides that present more than 60 variables.

Carmichael, 1994. Scientific American, 270:78-86

Introduction
Besides oral and intravenous routes of intoxication, human beings can be exposed to cyanotoxins through inhalation. It has been already confirmed that this toxin can reach the lungs, and its distribution after oral and intratracheal administrations was well described by Ito et al (2000, 2001). However, there are no reports analyzing MC effects on lung function. The aim of this study was to analyze pulmonary mechanical and histological profiles in mice after intratracheal administration of MCLR.

Materials and Methods

Swiss (n=56)

Saline (90 L, i.t.)

MC-LR (40 g/kg, i.t.)

Ctrl (n=6)

2h (n=11)

8h (n=11)

24 h (n=10)

48 h (n=9)

96 h (n=9)

Materials and Methods

At 2, 8, 24, 48 and 96 hours after the instillation, 1-2 animals of C group and 9-11 animals of Cyanobacteria group were sedated (diazepam 1 mg, i.p.), anesthetized (pentobarbital sodium 20 mg/kg, i.p.), tracheotomized and mechanically ventilated (V = 1.0 mL/s and V = 0.2 mL). The thorax was opened and the positive end-expiratory pressure (PEEP) value was determined in each animal according to the lung elastic recoil pressure at functional residual capacity (FRC). PEEP applied was around 2 cmH2O.

Materials and Methods

Airflow and transpulmonary pressure were measured. Volume was obtained by integration of the flow signal. All signals were conditioned, amplified and passed through low-pass 8-pole Bessel filters, analog-to-digital converted, and stored on a computer. All data were collected using LABDAT software. The pulmonary mechanical parameters were computed by the end-inflation occlusion method (Figure 1).

Materials and Methods

1.25

1,25

Flow (mL.s-1 )

0 0

P1 = Pmax Pi P2 = Pi Pel Ptot = P1 + P2

Pmax,L Pmax,L Pi,L P1,L P1,L P2,L P2,L Pel ,L Pel,L

- .25 1 -1,25

V (mL)

0.25

0.0

2 PL (cmH2O)

Est = Pel / VT Edyn = Pi / VT E = Edyn Est

Time (s)

Figure 1: End-Inflation Occlusion Method. From top to bottom - flow, volume (V) and transpulmonary pressure (PL). Airway occlusions were performed at the point indicated by the first arrow on the volume tracing. After 5 s, occlusions were released (second arrow).

Materials and Methods

At the end of the experiments, 6 mice from Ctrl and 4-7 from cyanobacteria groups were exsaguinated and the lungs were removed en bloc. The lungs were quickly-frozen by immersion in liquid nitrogen and fixed with Carnoys solution at 70C for 24 h. The slices were stained with H&E and histological analysis was performed using the point-counting technique.

Statistical Analysis
Normality distribution and homogeneity of variances were

tested (Kolmogorov-Smirnov test, with Lilliefors correction and Levene test, respectively). If both conditions were satisfied, one-way ANOVA was used. In the negative case, the nonparametric Kruskal Wallis ANOVA on Ranks was used. Bonferroni t-test was applied for multiple comparisons. In all tests the significance level was set at 5%. Statistical analysis was done with SigmaStat 3.11 statistical software package (Systat Software Inc., San Jose, CA, USA).

Results
100

20

Est (cmH2O/mL)

E (cmH2O/mL)

80 60 40 20 0

16 12 8 4 0

Ctrl

2h

8h

24 h

48 h 96 h

Ctrl

2h

8h

24 h

48 h 96 h

Figure 2: Static elastance (Est) and elastic component of viscoelasticity ( E) in control (Ctrl) and cyanobacteria groups 2, 8, 24, 48 and 96 hours after the instillation with saline or MC-LR, respectively. Values are median, 10, 25, 75 and 90% percentiles. *Significantly different from Ctrl group (p<0.05).

2,0

P1 (cmH2O)

P2 (cmH2O)

1,5 1,0 0,5 0,0 Ctrl 2h 8h 24 h 48 h 96 h

3 2 1 0

Ctrl

2h

8h

24 h 48 h 96 h

Ptot (cmH2O)

5 4 3 2 1 0 Ctrl

2h

8h

24 h

48 h 96 h

Figure 3: Resistive (P1), viscoelastic/inhomogeneous (P2) and total (Ptot) pressures variations of the lung in control (Ctrl) and cyanobacteria groups 2, 8, 24, 48 and 96 hours after the instillation with saline or MC-LR, respectively. Values are median, 10, 25, 75 and 90% percentiles. *Significantly different from Ctrl group (p<0.05).

Results
100 80
Ctrl 2h 8h 24 h 48 h 96 h

Area (%)

60 40 20 0

Normal

Collapsed

Figure 4: Morphometric data as percentage of normal and collapsed areas in control (Ctrl) and cyanobacteria groups 2, 8, 24, 48 and 96 hours after the instillation with saline or MC-LR, respectively. Values are means + SEM. *Significantly different from Ctrl group (p<0.05).

Results
60

Cellularity (%)

50 40 30 20 10 0

Ctrl 2h 8h 24 h 48 h 96 h

** **
MN PMN TOTAL

Figure 5: Polymorphonuclear (PMN), mononuclear (MN) and total cells (TOTAL) in pulmonary tissue in control (Ctrl) and cyanobacteria groups 2, 8, 24, 48 and 96 hours after the instillation with saline or MC-LR, respectively. Values are means + SEM. *Significantly different from Ctrl group (p<0.05).

Results
A B C

100 m

100 m

100 m

100 m

100 m

100 m

Figure 6: Photomicrographs of pulmonary parenchyma (200x). A, control group; B, C, D, E, F, animals sacrificed at 2, 8, 24, 48 and 96 hours, respectively, after the instillation of a sub-letal dose (40 g/kg) of MC-LR.

Results
A B C

Figure 6: Photomicrographs of pulmonary parenchyma (200x). A, control group; B, C, D, E, F, animals sacrificed at 2, 8, 24, 48 and 96 hours, respectively, after the instillation of a sub-letal dose (40 g/kg) of MC-LR.

Conclusions
Intratracheal exposure to MC-LR led to a biphasic increase in elastic and viscoelastic components of lung mechanics, which was characterized by an early increase (2 h) followed by normalization at 24 h and a late compromise of lung function at 48 h.

The kinetic of the observed data suggests an initial direct insult followed by a secondary bout probably resulting from the recirculation of MC-LR, which has been already described in human beings.