Effects Of Restraint Position On The Changes Of Diaphragmatic Mechanical Characteristic In Rats

Objective To observe the effects of restraint position on the changes of diaphragmatic mechanical characteristic of rats and the relationship with the changes of respiratory dysfunctions, try to elucidate the roles of diaphragmatic NO in the changes of diaphragmatic mechanical characteristic during restraint position of rats.Methods Rat models of restraint position and cecal ligation and puncture(CLP) were established respectively. Experiment in the effects of restraint position on the changes of diaphragmatic mechanical characteristic of healthy rats was divided into control (C) group, restraint position 12h (R12h) group and restraint position 24h (R24h) group. Experiment in the effects of restraint position on the changes of diaphragmatic mechanical characteristic of CLP rats was divided into SHAM group, CLP12h group, CLP18h group and CLP12h+restraint position 1h (CLP+R) group. The parameters of respiratory functions were recorded as follows: respiratory rate (RR), tidal volume (Vt), minute ventilation (MV), and inspiratory time relative to total respiratory time (Ti/Ttot). The parameters of diaphragmatic mechanical characteristic were assessed as follows: peak twitch tension (Pt), maximum tetanic tension (Po), time to peak contraction (CT), half relaxation time (RT1/2), maximal rise velocity of tension(+DT/dtmax), maximal fall velocity of tension(-DT/dtmax).The force-frequency relationship and fatigue resistance. Pt, Po and force-frequency relationship were measured again after the administration of L-NNA. Serum NO level were measured by spectrophotometry. The expressions of nNOS and iNOS mRNA in diaphragm were detected with RT-PCR.Results Effects of restraint position on the changes of diaphragmatic mechanical characteristic of healthy rats The results were as follows:①R R, Vt and MV were significantly decreased (P<0.01), Ti/Ttot was increased (P<0.01) in the early stage of restraint position. Under this experimental condition, Ti/Ttot was increased (P<0.05) and RR, MV showed downtrend (P>0.05), and sigh-like breathing was observe in R24h group (vs R12h group).②C ompared with control group, Pt was significantly decreased(P<0.01), force-generating capacity in force-frequency relationship was significantly reduced only in 10Hz and 20Hz (P<0.05) in R12h group, and Pt, Po, +DT/dtmax and -DT/dtmax were significantly decreased (P<0.01), force-generating capacity over the full range of the force-frequency relationship was reduced (P<0.01) in R24h group. There were no differences in CT, RT1/2 among the three groups (P>0.05). Absolute force levels during fatiguing stimulation paralleled the changes in the force-frequency relationship, but there were no differences in the diaphragm fatigue resistance if the force loss was normalized to its initial level (P>0.05). Pt (not Po)was increased after L-NNA administration in three groups (P<0.05), Force-frequency relationship after L-NNA administration was reduced in R24h group (P<0.01 vs C group), but the effect of L-NNA was no difference among the three groups (P>0.05).③NO in serum was increased significantly during the restraint position of rats(P<0.01).④Diaphragmatic nNOS mRNA expression was upregulated significantly during the restraint position (P<0.01); but there was little or no iNOS mRNA expression among the three groups (P>0.05).⑤Compared with control group, local diaphragmatic fibers were disorganized in R12h group, and atrophied and dissolved in R24h group. Effects of restraint position on the changes of diaphragmatic mechanical characteristic of CLP rats The results were as follows:①C LP rats showed significant respiratory dysfunctions, while restraint position with CLP rats could lead to severe respiratory dysfunctions with a transient compensatory period (about 60min). Such as respiratory frequency decreased progressively, sigh-like breathing frequently, and contradictory thoracic and abdominal movement, convulsion and mouth breathing appeared.②P t, Po, +DT/dtmax and -DT/dtmax were significantly declined in CLP12-18h group (P<0.01). And these parameters (except Po) were further declined in CLP12h+R group (P<0.01). CT and RT1/2 in CLP12h group had no significant change (P>0.05) (vs SHAM group), but in CLP18h group, CT was declined and RT1/2 was increased (P<0.01). Compared with CLP12h or CLP18h group, CT and RT1/2 in CLP12h +R group were significantly declined (P<0.01).③F orce-generating capacity over the full range of the force-frequency relationship was significantly declined in CLP12-18h group (P<0.01). And force-generating capacity of the force-frequency relationship in 10Hz ~ 60Hz in CLP12h+R group was further declined (P<0.01).④Absolute force levels during fatiguing stimulation paralleled changes in the force-frequency relationship, and there were differences in 0s～45s in the diaphragm fatigue resistance once the force loss was normalized to its initial level compared with SHAM group(P<0.05).⑤Both Pt and Po were significantly increased after L-NNA administration in four groups (P<0.01). Force-generating capacity in force-frequency relationship after L-NNA administration was significantly increased in CLP12-18h group (P<0.01). The effect of L-NNA was no difference between CLP12h and CLP12h+R group (P>0.05), but was different from CLP18h group (P<0.01).⑥NO level in serum was increased significantly in CLP12-18h group (P<0.01). NO level in serum in CLP12h+R group was lack of statistical significance with those of CLP12h and CLP18h group (P>0.05).⑦D iaphragmatic iNOS mRNA expression was upregulated significantly in CLP12-18h group (P<0.01). There were no differences in iNOS mRNA expression between CLP12h+R and CLP12h groups (P>0.05), but iNOS mRNA expression in CLP12h+R group was significantly lower than that in CLP18h group (P<0.01).⑧Compared with control group, diaphragmatic fibers were disorganized, atrophied and dissolved in CLP12h group, CLP18h and in CLP12h +R group.Conclusion In healthy rats restraint position induced respiratory dysfunctions, diaphragmatic contractility decrease and the upregulation of nNOS mRNA expression in diaphragm as well as the NO level increase in serum. Restraint position of CLP rats led to diaphragmatic contractility decrease sharply, severe respiratory dysfunctions and the upregulation of iNOS mRNA expression in diaphragm as well as the NO level increase in serum significantly. NO from diaphragma or circulation comtributed to diaphragmatic contractility decrease and respiratory dysfunctions. Diaphragms in healthy rats with free or restraint position have a very strong reserve capacity, which comtributed to the nonlinear relationship between respiratory dysfunctions and diaphragmatic contractility decrease.