Role of insulin in oleic acid-induced acute lung injury in rat model

Animals, anaesthesia and recording procedure

All the animal experiments were performed after approval from the Institute Ethical Committee of Institute of Medical Sciences, Banaras Hindu University, Varanasi, India (Approval letter no. Dean/2016/CAEC/621). OA-induced ALI rat model which mimics ALI in human was used for the study.^[4] Adult male albino rats of Charles-Foster strain weighing (175–225 g) were selected for the study. Animals were kept in a temperature (25 ± 0.5°C), humidity (50% RH) and light controlled room (12 h:12 h, light-dark period).^[3] Commercially available rat normal pellet diet (Hindustan Lever, Mumbai, India) and water were given ad libitum.^[14,15] Urethane (1.5 g/kg body weight i.p.) was used to anaesthetise the animal.^[3] After anaesthetising the rats, tracheal, jugular vein and carotid artery cannulation were done to keep the respiratory tract patent, saline/ drug administration and recording of blood pressure (BP), respectively. The respiratory excursions were recorded on a chart recorder through the force-displacement transducer attached with the help of thread secured to the skin over the xiphisternum to determine respiratory frequency (RF). Carotid artery was connected with pressure transducer for recording BP to determine the mean arterial pressure (MAP). ECG potentials were recorded by needle electrodes connected to bioamplifier using limb lead II configuration. All the recordings were made on a computerised chart recorder (Lab Chart 7, AD Instrument, Australia). Heart rate (HR) was calculated from R to R interval.^[4,16]

Experimental design

After dissection and cannulation, the animals were allowed to stabilise for 30 min.^[3] The rats were randomly divided into three groups.

In Group I (saline control group, n = 6), after initial recording, saline (75 μL) was injected through the jugular vein and the changes in RF, HR and BP were recorded at 2 min and then at every 15 min up to 120 min.^[3] These animals served as time-matched control group.

In Group II (OA group, n = 6), after initial recording, 75 μL OA was injected through jugular vein and the changes in RF, HR and BP were recorded at 2 min and then at every 15 min up to 120 min or till the death of the animal, whichever was earlier.

In Group III (insulin + OA group, n = 6) after initial recording, insulin 0.2 unit (0.5 U/Kg of body weight) was injected intravenously through jugular vein and the changes in RF, HR and BP were recorded.^[13] Fifteen minutes after insulin pre-treatment, OA 75 μL was injected and the recordings were taken as mentioned for Group II. At the end of each experiment, the lungs were excised. Lung of one side was kept for the estimation of pulmonary water content and other side was sent for histological examination.

Determination of pulmonary water content

The pulmonary water content was determined by physical method as described earlier.^[2,4,17] Briefly, at the end of each experiment, the lungs were excised, one lung was preserved in formal saline for histological examination and the other was weighted and dried to a constant weight in an electric oven (at 90°C for 48 h). The difference between wet weight and dry weight was calculated to determine the water content.^[4,17] This parameter was used as an indicator for pulmonary oedema developed during experiment.

PaO₂/FiO₂

The carotid arterial blood sample was collected into heparin rinsed syringe after 15 min of OA/saline injection in all groups.^[16] The blood sample was subjected to arterial blood gas analysis (Roche OMNI gas analyser) to determine the PaO₂/FiO₂ ratio, where PaO₂ is partial pressure of oxygen in arterial blood and FiO₂ is fraction of oxygen in the inspired air (FiO₂ considered as 0.21).

Histology of lungs

The lung tissue was fixed in formal saline and was subjected to standard histological protocol and stained with haematoxylin and eosin for microscopic examination.^[18]

Drugs and solutions

Urethane was obtained from Sigma-Aldrich Inc., St. Louis, USA, and prepared in double-distilled water. OA was obtained from Hi-Media Laboratories Pvt. Limited, Mumbai. Insulin (2 IU) was obtained from Gland Pharma Ltd., Hyderabad.

Analysis of data

The changes in RF, HR and MAP were expressed as % of initial. The data were pooled and mean ± standard error of mean (SEM) were calculated. Sigma Plot 10.0 and MS Excel software were used for the statistical and graphical analysis. The comparisons among the groups were performed using one-way ANOVA (Kruskal–Wallis test) followed by post hoc test (Student’s Newman–Keuls test) to find out pairwise differences in mean values. Wilcoxon paired sample test was used to test significant changes at two time points that is, initial to 30 min within the groups. Kaplan–Meier’s survival curves and log rank (Mantel–Haenszel) test were used for comparing the survival time.^[19] P < 0.05 was considered statistically significant.

Effect of saline on pulmonary and cardiac parameter

Saline-treated animals were taken as control group. After stabilisation, recording of RF, HR and BP was done. There was no significant change observed in the RF, HR and MAP [Figures 1-3] throughout the recording time (120 min) in saline treated rats. All the animals in this group survived for the entire observation period [Figure 4]. The PaO₂/FiO₂ was 454.67 ± 3.87 and pulmonary water content in this group was 77.74 ± 0.83% [Figure 5]. Histological examination of lungs exhibited the well-aerated alveoli which form the parenchyma of the lung. Alveoli were lined by a simple squamous epithelium [Figure 6a and b].

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Figure 1:

Effect of OA (75 µl) on RF (% of initial) in rats without or with insulin pre-treatment. Insulin was injected 15 min before injection of OA. RF at ‘0’ min (time of OA injection) was taken as 100%. Mean ± standard error of mean values were obtained from six different experiments in control, OA and insulin + OA groups. Changes in the RF in insulin + OA group were significantly different from OA group (P<0.05; one-way ANOVA). Insulin + OA = Insulin + OA-treated group. OA: Oleic acid, RF: Respiratory frequency.

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Figure 2:

Effect of OA (75 µL) on HR (% of initial) in rats without or with insulin pre-treatment. OA was injected at ‘0’ min. Insulin was injected 15 min before injection of OA. HR at ‘0’ min (time of injection of OA) was taken as 100%. Mean ± standard error of mean values were obtained from six different experiments in both insulin + OA group and in OA group. Changes in the insulin + OA group were similar to OA group (P<0.05; one-way ANOVA). Insulin + OA = Insulin + OA-treated group. OA: Oleic acid, HR: Heart rate.

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Figure 3:

Effect of OA (75 µL) on MAP (% of initial) in rats with or without insulin pre-treatment. OA was injected at ‘0’ min. Insulin was injected 15 min before injection of OA. MAP at ‘0’ min (time of injection of OA) was taken as 100%. Mean ± standard error of mean values were obtained from six different experiments in both insulin + OA group and in OA only group. Changes in the insulin + OA group were similar to OA group except at 2 min after OA injection (P<0.05; One-way ANOVA). Insulin + OA = Insulin + OA-treated group. OA: Oleic acid, MAP: Mean arterial pressure.

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Figure 4:

Kaplan–Meier’s survival plot for control, OA and insulin + OA groups. Overall survival in insulin + OA group was similar to OA group. Insulin + OA = Insulin + OA treated group, OA = Oleic acid.

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Figure 5:

Pulmonary water content (as % of wet lung tissue) in saline, OA and insulin + OA groups. Each bar depicts the Mean ± standard error of mean values obtained from six different experiments in each group. The asterisk (*) indicates significant difference from saline treated (control) group (P<0.05). Insulin + OA = Insulin + OA-treated group. OA: Oleic acid.

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Figure 6:

Photomicrograph of rat lung in saline control (a and b); oleic acid treated (c and d); insulin + oleic acid (e and f) groups are shown. (Magnification = ×400. “↖” [thick arrow] depicts alveolar damage, “ϟ” depicts infiltration of inflammatory cells and “↘” [thin arrow] interalveolar septa, “red arrow” depicts endothelial lining of blood vessels and “yellow arrow” depicts intravascular congestion).

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OA produced ALI

In OA group, after OA (75 μL) administration, there was a significant increase in RF (twice the initial, P < 0.05) by 30 min followed by progressive fall in RF [Figure 1]. By 90 min, respiration stopped leading to death of all the animals. The mean survival time in this group was 69.5 ± 3.55 min [Figure 4]. OA administration produced an immediate decrease in HR (43% of initial, P < 0.05) and MAP (about 45% of initial, P < 0.05; Figures 2 and 3) followed by recovery (about 23% of initial) by 30 min and then progressive decrease till death of the animals [Figure 4]. The mean ± SEM of PaO₂/FiO_{2 (}250.40 ± 9.03) was significantly lesser (P < 0.05) and pulmonary water content (83.91 ± 0.44%) was significantly increased (P < 0.05) as compared to control group [Figure 5]. Histological examination of lungs showed multifocal and heterogeneous lung injury, with interstitial oedema and infiltration of leucocytes leading to thickening of the alveolar septa. Intravascular congestion and capillary endothelial injury were apparent. The entire lung architecture was destroyed in certain areas and the alveolar space was found to be reduced [Figure 6c and d].

Effect of insulin pre-treatment on OA-induced ALI

The initial tachypnoea, as observed in OA only group, was not seen in insulin pre-treated (insulin + OA) group. The RF was maintained at good level (92% of initial, P < 0.05) till 30 min [Figure 1]. Then, there was progressive fall in RF till death of the animal and mean survival time in this group of animals was 66.83 ± 1.28 min, similar to OA only group (Figure 4; Kaplan–Meier’s survival plot, log rank test, P= 0.14,). HR and MAP showed significant decrease (about 20%, P < 0.05) followed by recovery and then progressive decrease till death of the animal [Figures 2 and 3]. The mean ± SEM of PaO₂/FiO₂ was 275.86 ± 2.44 and pulmonary water content 83.33 ± 0.53%, respectively, and was similar to OA group (250.40 ± 9.03 and 83.91 ± 0.44%; Figure 5). Histological examination of lungs exhibited multifocal and heterogeneous injury in these rats also. However, inflammatory cells seen in interalveolar septa and alveoli were less in number (per field of histological slide examination) as compared to OA group. Thickening of the alveolar septa in this group was also less as compared to OA group. Intravascular congestion similar to OA group was seen in this group also but injury to the endothelial lining of blood capillaries was not observed in this group of animals [Figure 6 e and f].