Corticosteroid effects on ventilator-induced diaphragm dysfunction in anesthetized rats depend on the dose administered

Abstract

Background: High dose of corticosteroids has been previously shown to protect against controlled mechanical ventilation (CMV)-induced diaphragmatic dysfunction while inhibiting calpain activation. Because literature suggests that the calpain inhibiting effect of corticosteroid depends on the dose administered, we determined whether lower doses of corticosteroids would also provide protection of the diaphragm during CMV. This may be important for patients undergoing mechanical ventilation and receiving corticosteroids.

Methods: Rats were assigned to controls or to 24 hours of CMV while being treated at the start of mechanical ventilation with a single intramuscular administration of either saline, or 5 mg/kg (low MP) or 30 mg/kg (high MP) of methylprednisolone.

Results: Diaphragmatic force was decreased after CMV and this was exacerbated in the low MP group while high MP rescued this diaphragmatic dysfunction. Atrophy was more severe in the low MP group than after CMV while no atrophy was observed in the high MP group. A significant and similar increase in calpain activity was observed in both the low MP and CMV groups whereas the high dose prevented calpain activation. Expression of calpastatin, the endogenous inhibitor of calpain, was decreased in the CMV and low MP groups but its level was preserved to controls in the high MP group. Caspase-3 activity increased in all CMV groups but to a lesser extent in the low and high MP groups. The 20S proteasome activity was increased in CMV only.

Conclusions: Administration of 30 mg/kg methylprednisolone during CMV protected against CMV-induced diaphragm dysfunction while 5 mg/kg was more deleterious. The protective effect is due mainly to an inhibition of the calpain system through preservation of calpastatin levels and to a lesser extent to a caspase-3 inhibition.

Figure 1

Diaphragm force generation. Upper panel: Force-frequency relationship in the diaphragm of controls (C, open rectangles, n = 9), controlled mechanical ventilation animals treated either with saline (CMV, closed triangles, n = 9) or with 5 mg/kg methylprednisolone (MP-5, open circles, n = 7) or 30 mg/kg methylprednisolone (MP-30, closed circles, n = 6). Maximal tetanic tension in the diaphragm of C, CMV, MP5 and MP30 groups. Values are means ± SD. *p < 0.001 CMV and MP-5 vs others, #p < 0.01 CMV vs C and MP-30, ++p < 0.01 MP-5 vs MP-30, +p < 0.05 MP-5 vs MP-30.

Figure 2

Diaphragm cross-sectional area of the different fiber types. Diaphragm cross-sectional area (CSA) of the type I, IIa and IIx/b fibers in the controls (C, n = 6) and controlled mechanical ventilation animals treated either with saline (CMV, n = 6) or with 5 mg/kg methylprednisolone (MP-5, n = 6) or 30 mg/kg methylprednisolone (MP-30, n = 5). Values are means ± SD. *p < 0.05 vs C and MP-30, #p < 0.05 vs CMV, +p < 0.01 vs C.

Figure 3

Western blot analysis of talin. Upper panel: A representative western blot for the analysis of intact and cleaved talin (BDP) in the diaphragm of controls (C, n = 6) and controlled mechanical ventilation with saline injection (CMV, n = 9) or with 5 mg/kg methylprednisolone (MP-5, n = 7) or 30 mg/kg methylprednisolone (MP-30, n = 5). Lower panel: Mean densitometric values for talin degradation by calpain. Values are shown as the ratio of degraded talin to intact protein and are expressed as means ± SD, *p < 0.05 vs others.

Figure 4

Western blot analysis of calpastatin. Upper panel: A representative western blot for calpastatin and α-tubulin in the diaphragm of controls (C, n = 6) and controlled mechanical ventilation with saline injection (CMV, n = 8) or with 5 mg/kg methylprednisolone (MP-5, n = 7) or 30 mg/kg methylprednsiolone (MP-30, n = 5). Lower panel: Densitometric values for calpastatin corrected for α-tubulin. Values are means ± SD, *p < 0.05 vs others.

Figure 5

Western blot analysis of αII-spectrin. Densitometric values for αII-spectrin degradation by caspase-3. Values are shown as the ratio of degraded αII-spectrin to intact protein and are expressed as means ± SD, *p < 0.01 vs C, #p < 0.05 vs CMV.