Enliven: Journal of Anesthesiology and Critical Care Medicine

Rationale: The mechanical ventilation may aggravate the severe acute lung injury and trigger a systemic inflammatory response. The pathophysiology evolves Biotrauma, meaning that the over distension and the cyclical opening/collapse of structures on the lung during ventilation with high PIP associated with low PEEP, results in excessive activation of nuclear factor-{kappa}Beta (NF-κB), a regulator of the inflammatory response, that exacerbates the local response and becomes it systemic. The magnesium, a modulator of signaling pathways of the inflammatory cascade, deserves investigation in inhibiting the NF-κB activation.

Objectives: Investigate experimentally the effect of hypermagnesemia to inhibit the activation and migration of NF-κB from cytoplasm to nucleus in cells of lungs injured by mechanical ventilation.

Methods: Two experimental groups of 15 Sprague-Dawley rats each, one receiving 1.25 mL/kg/h of a 10% MgSO₄ solution, other receiving equal volume of saline, both under mechanical ventilation with VT of 40 mL/kg, PEEP of 3 cmH₂O, PIP of 35 cm H₂O, RR of 40 cycles per minute. Lungs were excised after 50 minutes of experimental interventions and prepared for further immunohistochemical analysis.

Measurements and Main Results: ANOVA shown no differences when compared the NF-κB activation values in the cytoplasm (712.44 ± 253.86) and nucleus (974.27 ± 344.59) of cells in MgSO₄ group with the respective values in cytoplasm (653.99 ± 272.98) and nucleus (952.29 ± 346.68) of cells in Saline group (F_{[1, 28]} = 0.90, p > .05).

Conclusions: There was no inhibitory effect of magnesium on the activation of NF-kB nor on its expression through migration to the nucleus.

Introduction

The mortality rate associated with severe acute lung injury exceeds 50%, and this is relate to the multiple organ failure than to hypoxia by respiratory failure [1-5]. Mechanical ventilation can aggravate [6] or even be among the causes of acute lung injury [7], and trigger a systemic inflammatory response.

Ventilator Induced Lung Injury (VILI) do not differ in essence of Acute Respiratory Distress Syndrome (ARDS) [8-10], with edema containing red cells, neutrophils, serum proteins and surfactant, pointing to the increase of permeability of alveolar-capillary barrier [11-18], which pathophysiology involves “biotrauma” [4,19-24]. High pressures during mechanical ventilation potentially stresses the pulmonary architecture, leading to (by over-distension) a loss of integrity of plasmatic membrane in alveolar cells with decompartmentalization into the alveolar space and release of intracellular cytokines to the interstitium and systemic circulation [25]. The cyclical opening/collapse of pulmonary structures release (by mechanical transduction) cytokines and chemokines that induce an excessive activation of nuclear factor-{kappa}Beta (NF-kB) [4,16,26-37].
Some strategies aiming to block the NF-κB activation are based on inhibition of its migration for the nucleus [38-41]. Angiotensin-converting enzyme-2 [42] and the activator protein (AP)-1 [43], e.g., have been tested in the lipopolysaccharide–induced lesion model of the pulmonary microvascular endothelial cells, but not in the VILI model. Since Trendelenburg (1912) accepted, and Rosello and Pla (1936) demonstrated, the effect of magnesium as a potential bronchodilator, its low oral intake has been related to airway hyper-reactivity, and oral supplementation apparently helps to control asthma in pediatric patients [44]. Magnesium has a potential effect in inhibiting inflammation [45].

Alveolar macrophages of hypomagnesemic rats show high serum levels of IL 1-β e IL 6, as well as induction of mRNA for IL 1-β and TNF-α [46], possibly explaining the bronchial hyperreactivity [47]. Moreover, hypomagnesemia is frequently observed in patients with higher risk to develop ARDS [48,49].

Magnesium sulphate (MgSO₄), useful in acute exacerbation of asthma at high doses [50], inhibits (in vitro) the secretion of the macrophage secretion of the inflammatory factor high mobility group box 1 (HMGB1) during experimentally induced sepsis [51], and can attenuates pulmonary oxygen toxicity [52]. The magnesium-related mechanism of inhibition of the NF-κB activation and its signaling pathway apparently involves the calcium channels regulation [53-55], especially the L-type calcium channels [56,57] and N-methyl-D-aspartate (NMDA) antagonist [57], with anti-inflammatory effect mediated by increase of the phosphoinositide 3-kinase/Akt (PI3K/Akt) activity [58].

This study aimed to investigate experimentally the effect of hypermagnesemia to inhibit the activation of NF-kB in lungs injured by mechanical ventilation.

Methods and Materials

This study was approved by the Review Board for human and animal studies of the Santa Casa de São Paulo School of Medicine.

Trial Design

Two experimental groups, composite by Sprague-Dawley rats randomly selected to receive high dose of magnesium sulfate (MgSO₄ Group), or equal volume of saline solution (Saline Group: controls), both under mechanical ventilation with harmful parameters. Their lungs were compared for the cytoplasm and nuclear values of activated NF-κB (obtained by immunohistochemical analysis) after exposition to the experimental condition (MgSO₄ or Saline).

Histopathology and Immunohistochemistry

The histopathologycal changes in lungs, useful for determination of the success in obtain VILI, are reversible, so its need be fixed during the experimental conditions that induce lesions [13], before euthanasia. After excised, the lungs may be fixed and conserved in 10% formalin for 48h, embedded in paraffin, and routinely processed. The lungs were stained with hematoxylin and eosin for routine histological analysis [59].

The expression of transcription nuclear factor-{kappa}Beta (NF-κB) p65 [21] was analyzed by Complex Streptavidina-Biotina-peroxidase (LSAB) Method. The coloration guarantee the identification on nucleus (NF-?B actived) and on cytoplasm.

The extension of the lesion and the intensity of the immunohistochemical expression were determinated by Stereologic Method [60].

Data for normal lungs (non-ventilated) of Sprague-Dawley rats were previously determinated, and revealed that usually exist a small Density (cells or points of fluid/mm²) of red cells, neutrophils and linfocites in the parenchyma, and perivascular fluid (Table 2).
All the procedures involved in histopathology and immunohistochemistry from the fixation of excised lungs, were performed by a pathologist expert in lung analysis from Department of Pathology of Santa Casa de Misericórdia de São Paulo.

Sample Size Determination and Statistical Analysis

No background information was found to estimate the standard deviation in the Sprague-Dawley population for values of the independent and dependent variables considered in this study: so, the Glass’ Effect Size (δ) remains 0.5 [61] for all outcomes. A high (0.9) intraclass correlation (ρ) is expected for data that are obtained of each subject is your own control. Indeed, this study involves two independent samples, with each group treated as a single sample in a pre-post design [61]. Thus, Critical Effect Size (Δ) is 0.75, and at least 11 (10+1) cases per group are necessary for a significance level of 5% for one-tailed parametric tests, and guarantee 90% power for Paired t-Test and Two-factor Analysis of Variance with repeated measures on one factor [61,62] and comparisons among treatment means by Fisher-lsd [62].

Subjects

Thirty four male Sprague-Dawley rats were necessary to compound the two experimental groups with a equal minimal size of 11 subjects, considering that the ventilated rats were randomly exposed to one of the two experimental conditions and that 4 rats data were discharged: 2 rats that died during induction of pulmonary lesions and 2 lungs not good for analysis. At end, did remain for analysis data of 15 rats in “MgSO₄ Group” and 15 rats in “Saline Group”.

For allocation, the author used a list of random numbers: even numbers: infusion of 10% MgSO₄ solution; odd numbers: infusion of saline solution.

Materials

JELCO^® catheter (16 gauge); Siemmens-900® ventilator; ampoules of 10% magnesium sulfate solution, thiopental sodium, saline solution, 10% formalin solution, provided by the Central Laboratory of Santa Casa de Misericórdia de São Paulo; kit NF-κB p50, United States Biological.

Procedures

– Jejum for 6h before anesthesia. The RR was registered at rest. Then, anesthesia with thiopental sodium (50 mg/kg) by via intra-peritoneal injection;
– Tracheal intubation for mechanical ventilation, remained at physiological parameters (V_T: 5 mL/kg; FiO₂: 21%; PEEP: 5 mmHg; RR: 40 cpm) during the surgical procedures: right jugular vein dissection and cannulation for intravenous infusions (I.V.); dissection of right carotid artery for laboratory tests;
– Obtaining of blood sample (2 mL) for baseline values of [Mg²⁺], PaCO₂, PaO₂ and SaO₂;
– Then, aiming to reach hypermagnesemia, magnesium was infused in a high dose (1,25 mL/kg/h of a 10% MgSO₄ solution) for rats numbered with even numbers, and an equivalent volume of saline for the other rats;
– At the start of continuous I.V. infusion, the mechanical ventilator was adjusted to harmful values: V_T = 40 mL/kg; PEEP = 3 cm H₂O, PIP = 35 cm H₂O; RR = 40 cpm, remained until the end of infusion, i.e., 50 minutes.
– After the end of the infusion, new blood samples were obtained for serum [Mg²⁺], PaCO₂, PaO₂ and SaO₂ determinations.
– Then, infusion in right inner jugular vein of 20 mL of saline, followed by 20 mL of 10% formalin solution for the microscopy fixation of the lungs, that were then excised en bloc;
– At last, euthanasia with I.V. infusion of thiopental sodium, 50 mg/kg in bolus.

Conclusion

It is concluded that, for the subjects of this study, there were no inhibitory effect of high dose of magnesium on the activation of NF-kB neither on its expression through migration to the nucleus. In respect to its role on the systemic inflammatory response and on failure of multiple organs, both would not have been avoided in these rats. About local (pulmonary) inflammatory response, seems that magnesium was the responsible by avoiding interstitial edema.

Results and Discussion

Efficacy in Control of Potentially Intervenient Variables

A post hoc analysis of variance for multiple comparisons between MgSO₄ Group (N = 15) and Saline Group (N = 15) means (Table 1) revealed (considering each subject as your own control) that these two groups were similar for these means when they were compared at baseline and at post-ventilation states of PaCO₂ (F_{[1, 28]} = 3.06, p > .05).

After mechanical ventilation, both the groups showed higher PaO₂ means (F_{[1, 28]} = 6.10, p < .05), without difference between them.

The MgSO₄ Group had higher SaO₂ mean after ventilation (F_{[1, 28]} = 5.14, p < .05) (Table 1).

When compared with normal lungs (N = 15) predetermined means (Table 2), both ventilated groups had higher means of inflammatory infiltrate (neutrophils and linfocites) (F_{[2, 42]} = 6.53, p > .05) and of red cells (F_{[2, 42]} = 7.95, p > .05), without differences between the groups, but the MgSO₄ Group did not develop interstitial edema (F_{[1, 42]} = 1.40, p > .05).

Paired t-test of comparisons between the serum [Mg²⁺] at base-line and after infusions (Table 1), reveals post-infusion hypermagnesemia in MgSO₄ Group (t = 4.130, df = 14, p < .05), and consume of magnesium in the controls (Saline Group) (t = 5.691, df = 14, p < .05).

Hypermagnesemia and the Blockage of NF-κB Activation

The effectiveness of inhibiting NF-κB activation and expression in lungs harmed by mechanical ventilation was analyzed through the comparison of the proportion of activated NF-κB in the cytoplasm and in the nucleus of the lungs of the subjects of experimental groups, reflecting the migration to nucleus for its expression.

Analysis of variance (two-way, repeated measures on one factor) reveal that activated NF-κB density means were higher in nucleus (974.27 ± 344.59) than in the cytoplasm (712.44 ± 253.86) in MgSO₄ Group lung cells, and also higher in nucleus (952.29 ± 346.68) than in the cytoplasm (653.99 ± 272.98) in Saline Group lung cells, without difference between groups (Table 3 and Table 4, Figure 1 and Figure 2).

Hematoxylinandeosinstain. 100x. A: Lungofthe non-ventilatedrat n. 6; B: Lungoftherat n. 15 (MgSO₄group), without perivascular edema; C: Perivascular/intersitialedema, rat n. 7 (Saline Group).

Hematoxylinandeosinstain. 100x. A: Lungofthe non-ventilatedrat n. 6; B: Lungoftherat n. 15 (MgSO₄group), without perivascular edema; C: Perivascular/intersitialedema, rat n. 7 (Saline Group).

Discussion

Although any significant difference among subjects of a same breed (Sprague-Dawley rats) was not expected before the experimental treatments, a post hoc analysis revealed that the MgSO₄ and Saline (control) groups, so that each subject, were comparable at the base line state. Moreover, in comparison with control group, data of experimental group revealed that the intravenous MgSO₄ infusion posology was enough to induce hypermagnesemia.

The histopathological analysis showed that there were success in the induction of lung damage with the mechanical ventilation parameters adopted in both groups, so that the effect of independent variable, probably, may not be suffered meddling of spurious variability: The isolation of the hypermagnesemia effect on the NF-κB activation was, seemingly, guaranteed.

The results obtained reveal that the NF-κB activation occurred during the induction of lung injury in both control and MgSO4 groups in a same extension, leading to conclusion that, in these Sprague-Dawley rats, MgSO₄ did not have effect on this activation. However, two findings deserved attention: the consumption of Mg²⁺ during lung damage induction; in the group that received MgSO₄, the lungs showed less perivascular edema.

The consumption of Mg²⁺ is not surprising, to the extent that is frequently observed in critically ill patients, and that Dedhia and Banks [48] and Bohmer [49] already had related hypomagnesemia as a higher risk of ARDS development. Maybe this consumption is related to the pathophysiologic mechanisms of acute lung injury, deserving future investigation.

About the perivascular edema, the lungs of the rats that received MgSO₄ showed, at mean, the same proportion of perivascular fluid as the non-ventilated subjects; those that received isotonic saline solution and developed hypomagnesemia post-VPM evolved with pulmonary edema. These results suggest a protective effect of magnesium on the formation of lung edema during pulmonary ventilation with potentially harmful parameters.

Whereas that in the VILI the edema imply fluidic overflow for the interstitial space, with alveolar flood partially due to high pressures over lung structures during alveolar recruitment [23,24], this finding deserves experimental investigation, particularly because the magnesium, that has a confirmed effect as an coadjuvant on control of acute asthma (edema has a relevant role in Asthma), may be involved in the tissular fluid homeostasis.

Acknowledgments

The authors thank to:
Fabíola Del Carlo Bernardi, MD, PhD, Pathologist, by histopathological and immunohistochemical analysis; Hugo Hideo Kunni, MD, who intermediated the Sprague-Dawley rats acquisition at the Laboratory of Animal Research of University of Campinas (UNICAMP), São Paulo, Brazil, and its transportation to the Unity of Experimental Surgical Technique (UTECE) of Santa Casa de São Paulo School of Medicine (FCMSCSP), São Paulo, Brazil.

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