Vol 8 (02) Feb ’18

International Journal of Livestock Research eISSN : 2277-1964 NAAS Score -5.36

Original Research

Histopathological and Scanning Electron Microscopy (SEM) of Paraquat

(PQ) induced Acute Lung Injury (ALI) in Experimental Rats
B. Haripriya1, M. Lakshman2* and V. Sudha3
1
M.Sc (Bio-Tech), Sri Padmavathi Mahila Viswa Vidhyalayam (SPMVV), Telengana, INDIA
2
Professor & HOD of Department of Veterinary Pathology and Officer-In-charge, Ruska Labs (Electron
Microscope Laboratory), P V Narsimha Rao Telengana Veterinary University (PVNRTVU), Telengana
State, INDIA
3
Veterinary Assistant Surgeon, Department of Animal Husbandry, Government of Telengana, INDIA

*Corresponding author: mekala_bry@yahoo.com

Rec. Date: May 25, 2017 04:01

Accept Date: Nov 11, 2017 14:52
Published Online: January 31, 2018
DOI 10.5455/ijlr.20170525040105

Abstract
The present study was aimed to know the histopathological and ultrastructural (SEM) alterations of lung
due to PQ (Herbicide) in male Albino wistar rats. The experiment was carried out at different time
intervals (24, 48, and 72 hrs). Histopathologically group 2 lung sections revealed increased thickness of
septa and collapsed alveoli. Tertiary bronchiolar epithelial cells showed mild to moderate hyperplasia
and focal areas inflammatory cells infiltration in peribronchiolar area. A dark inclusion like bodies and
mild fibrous tissue proliferation in peribrochiolar along with severe congestion was also observed. Few
bronchioles were shrunken. Scanning electron microscopy of group 2 lung slices showed irregular
surface area with hemorrhages in bronchiolar area. Specimens of 48 and 72 hours showed thickened
alveolar septa with mild to moderate fibrosis in peribranchiolar area.

Key words: ALI, Albino Wistar Rats, Histopatology and SEM, Paraquat, Toxicity

How to cite: Haripriya, B., Lakshman, M., & Sudha, V. (2018). Histopathological and Scanning Electron
Microscopy (SEM) of Paraquat(PQ) induced Acute Lung Injury (ALI) in Experimental Rats. International
Journal of Livestock Research, 8(2), 205-209. http://dx.doi.org/10.5455/ijlr.20170525040105

Introduction
Sustainable agricultural production and quality food security has always believed to be challenge in
growing population of the country. Pesticides and other chemicals are of precious inputs in sustaining
agricultural practices in Indian agricultural scenario. As a post green revolution which ensured the
205

quantity of production but quality which is remain unclear. Paraquat (PQ) is one of the highly toxic potent
herbicides promoted by the United States for use in Mexico to destroy marijuana plants in the year 1955.
Page

Frequent PQ poisoning incidents have become a severe public health issue all over world, especially in

Hosted@www.ijlr.org DOI 10.5455/ijlr.20170525040105

 Vol 8 (02) Feb ’18
International Journal of Livestock Research eISSN : 2277-1964 NAAS Score -5.36

Asian region. The paraquat (1,1′-dimethyl-4,4′-bipyridilium dichloride - PQ), is one of the most widely
used herbicides and holds a largest share of the global herbicide market till today. It is a non-selective
quaternary nitrogen herbicide, is commonly used as a desiccant and defoliant in a variety of crops all
around the world (Dasta, 1978; Bismuth et al., 1982&1990 and Raghu et al., 2013). For the past 60 years
PQ is considered a most toxic compound, which is classified as a moderately hazardous herbicide and
placed in poison class II by WHO (2009) due to its acute toxicity.

Materials and Methods

In the present study a total of 36 male albino Wistar rats weighing between 180-
240 g were procured from G. Pullareddy College of Pharmacy, Hyderabad. The experiment was
conducted after approval of IAEC (GPRCP/IAEC/07/17/01/PCL/AE-3-Rats-M-12). The rats were housed
in solid bottom polypropylene cages at Ruska Labs and were maintained under controlled environment
(Temperature 20-220C) and sterile rice husk was used as standard bedding material, all rats were
provided with standard pellet diet and deionized water ad libitum throughout the experimental period.
The experimental design adopted for the present study is shown in Table 1.

Table 1: The experimental design adopted for the present study

Group No. of Animals Treatment
Group-I 18 Basel diet
Group-II 18 Paraquat (I/P) single dose @24 mg/kbw
Group-II(a) 06 Paraquat (I/P) single dose @ 24 mg/kbw
Group-II(b) 06 Paraquat (I/P) single dose @ 24 mg/kbw
Group-II(c) 06 Paraquat (I/P) single dose @ 24 mg/kbw

The tissue samples of lung slices (1×1 cm3) were collected and fixed in 10% neutral buffer formalin
(NBF) soon after sacrifice. The samples were processed, sectioned (5μm) and stained with Hematoxylin
and Eosin (H&E) for histopathological examination as per the standard procedure (Luna, 1968). Soon
after sacrifice thin slices (1x1mm3) of lung were dissected and fixed in 2.5% gluteraldehyde (Sigma
Aldrich, USA) in 0.1M phosphate buffer (pH 7.3) and stored at 40C for 12 hours. And washed with
buffer, post fixed in 1% aqueous osmium tetroxide (Sigma Aldrich, USA) for 2 hours then dehydrated in
ascending grades of alcohol (Qualigens fine chemicals, Mumbai), and the dehydrated specimens were
subjected to vacuum desiccation for 45 minutes and mounted over stubs on the double sided carbon
conductivity tape and coated with Gold by using sputter coater (JEOL-JFC-1600) for 180 seconds. Later
specimens were observed under Scanning Electron Microscope (JEOL; JSM-5600, Japan).
206

Results and Discussion

Page

Hosted@www.ijlr.org DOI 10.5455/ijlr.20170525040105

 Vol 8 (02) Feb ’18
International Journal of Livestock Research eISSN : 2277-1964 NAAS Score -5.36

Gross Pathology
The rats were sacrificed at 24, 48 and 72 hrs time intervals of experiment and abnormalities were
recorded if any. In lung diffused hemorrhages were seen in all lobes. Small grey necrotic spots were also
observed on apical. On cut section blood tinged and oozed out. At 48 hrs of time interval one rat was
found dead where the diaphragmatic lobes, middle lobes of lungs showed severe haemorrhages and the
other part of lung showed emphysema (Fig.1). These observations were similar to the results of Hampson
and Pond, (1988) and Lalrautfela et al. (2014). These changes might be due to the toxic effects of PQ as
lung is the primary target organ (Lewis & Nemery, 1995 and Dinis-Oliveira et al., 2006).

Fig. 1: Showing 48 hrs treated Fig. 2: Photo micrograph of lung Fig. 3: Photo micrograph of lung
lung with mild to moderate showing fibrin deposition (H&E showing increased thickness of
congestion. 50µm) (24 hrs). septa, showing numerous round
cells and severe congestion
(H&E 100µm) (24 hrs).

Histopathology
Group two (24 hrs) lung sections revealed an increased thickness of septa, severe congestion along with
numerous round cell infiltrations was observed. A prominent dark dense inclusion like bodies with
vacuolar degeneration and fibrin deposition was also noticed (Fig. 2&3). The sections of 48 hrs treated
lung depicted the perivascular infiltration of inflammatory cells with numerous RBC’s giving red
hepatisation texture to the lungs. Majority sections showed proliferation of fibrous tissue which led to
shrinkage of bronchiole (Fig. 4&5). At 72 hrs intervals the treated lungs showed altered alveolar septa and
severe hemorrhages, congestion and hyperplasia of tertiary bronchiolar epithelial cells. Hyperplasia and
fibrosis of peribronchiole was the principle lesion in addition to deposition of exudates (Fig. 6). Theses
lesions might be due to the PQ induces mitochondrial damage of the alveolar cells. The similar changes
were explained by several authors (Dinis-Oliveira et al. (2006); Chang et al. (2009); Lalrautfela et al.
207

(2014); Zhang et al. (2014); Han et al. (2015) and Li et al.) in both sexes of Wistar rats in their
Page

experiments.

Hosted@www.ijlr.org DOI 10.5455/ijlr.20170525040105

 Vol 8 (02) Feb ’18
International Journal of Livestock Research eISSN : 2277-1964 NAAS Score -5.36

Fig. 4: Photo micrograph of lung
showing perivascular infiltration
of cells, red hepatisation and
fibrous tissue proliferation (H&E
100µm) (48 hrs)
Fig. 5: Photo micrograph of lung Fig. 6: Photo micrograph of lung
showing fibrous tissue showing peribranchiolar fibrosis
proliferation (H&E 50 µm) (48 and hyperplasia of brancheole
hrs). (H&E 50µm) (72 hrs).

Scanning Electron Microscopy

On the perusal of literature published data in relation to SEM in rats is not available, but it has been
documented in dog (TEM and SEM) by Hampson and S. M. Pond (1988). SEM slices of 24 hours lungs
showed hemorrhages in the septa with increased thickness and a note worthy feature of fibrosis in
peribroncheolar area during 48 and 72 hrs of experiment (Fig. 5&7). The proliferation of fibrous tissue at
the end of 24 hours were also observed, but during 48 and 72 hours it has reached to maximum extent
could be due to the activation of ROS and also TGF-β as the PQ is acting on the specific growth factors.
Hampson and S. M. Pond (1988) documented irregular alveolar walls, detached type I alveolar cells from
the basement membrane and accumulation of large numbers of erythrocytes and inflammatory cells
including macrophages which overlay the irregular walls of pneumocytes under SEM in paraquat exposed
canine.

208

Fig. 7: SEM of lung showing Fig. 8: SEM of lung showing Fig. 9: SEM of lung showing
haemorrhages in septa at x1500 proliferated fibrous tissue at proliferated fibrous tissue at
(24 hrs). x1000 (48 hrs) x2500 (72 hrs).
Page

Hosted@www.ijlr.org DOI 10.5455/ijlr.20170525040105

 Vol 8 (02) Feb ’18
International Journal of Livestock Research eISSN : 2277-1964 NAAS Score -5.36

Conclusion
The PQ is a proven compound to induce acute fibrosis in experimental rats. Grossly the lungs have
showed typical changes like red hepatization in a short period of experiment. Histopathologically, these
observations were supported by showing numerous RBC's in the interstitial area and infiltration of
inflammatory cells and proliferation of fibrous tissue along with increased alveolar septa in
peribronchiolar areas. Scanning electron observations have revealed the presence of hemorrhages and
proliferating fibrous tissue at various degrees. These observations conclude that the PQ is a potential
compound to induce acute fibrotic changes in experimental rats.

References
1. Dasta, JF. 1978. Paraquat poisoning: A review American Journal of Health-System Pharmacy 35 (11):
1368-1372.
2. Bismuth, C, Garnier, R, Baud, FJ, Muszynski,J and Keyes, C. 1990. Paraquat poisoning. Drug
Safety 5 (4): 243-251.
3. Raghu, K, Mahesh, V, Sasidhar, P, Reddy, PR, Venkataramaniah, V. and Agrawal, A.2013.Paraquat
poisoning: A case report and review of literature. Journal of family and community medicine 20 (3):
198-200.
4. Hampson, EC and Pond, SM. 1988. Ultrastructure of canine lung during the proliferative phase of
paraquat toxicity. British journal of experimental pathology 69 (1):57.
5. Lewis, CP. and Nemery, B. 1995.Pathophysiology and biochemical mechanisms of the pulmonary
toxicity of paraquat.vol.10, Marcel Dekker, New York, NY,USA.
6. Lalruatfela, PL, Saminathan, M. Ingole, RS, Dhama, K. and Joshi, MV. (2014). Asian Journal of
Animal and Veterinary Advances 9:524-542.
7. Zhang, Z, Ding, L, Wu, L, Xu, L, Zheng, L. and Huang, X. 2014.Salidroside alleviate sparaquat-
induced rat acute lung injury by repressing TGF-β1 expression. International journal of clinical and
experimental pathology 7 (12):8841-8847.
8. Dinis-Oliveira, RJ, Duarte, JA, Sanchez-Navarro, A, Remiao, F, Bastos, ML. and Carvalho, F,
2008.Paraquat poisonings: mechanisms of lung toxicity, clinical features, and treatment. Critical
reviews in toxicology38 (1): 13-71.
9. Han, J, Ma, D, Zhang, M, Yang, X. and Tan, D. 2015. Natural antioxidant betanin protects rats from
paraquat-induced acute lung injury interstitial pneumonia. Bio Med Research International: 1-9.

209
Page

Hosted@www.ijlr.org DOI 10.5455/ijlr.20170525040105