Inflammation ( # 2016) DOI: 10.1007/s10753-016-0397-0

ORIGINAL ARTICLE

Effects of Theophylline with Methylprednisolone Combination Therapy on Biomechanics and Histopathology in Diaphragm Muscles of Rats Nureddin Yuzkat,1,6 Ismail Kati,2 Yasemin Isik,3 Servet Kavak,4 Ugur Goktas,1 and Nurettin Cengiz5

Abstract—The purpose of this study was to investigate the effects of theophylline and methylprednisolone on the mechanical response and histopathology of hemidiaphragm muscle in rats. In the current study, we aimed to investigate the effects of theophylline and methylprednisolone, which are frequently used in clinics and which have different effects on the respiratory system and on the biomechanics and histopathology of the diaphragm muscle. The study included four groups of rats. Group T received 1 mg/ kg of intraperitoneal theophylline, group M received 2 mg/kg of intraperitoneal methylprednisolone, group TM received 1 mg/kg of intraperitoneal theophylline plus 2 mg/kg of intraperitoneal methylprednisolone, and group K received of 1 mL intraperitoneal isotonic solution (of 0.9 % NaCl). The medications were continued for 7 days in each group. The rats underwent cervical dislocation under anesthesia on the eighth day, and their diaphragm samples were extracted. The left hemidiaphragm was used for the investigation of biomechanical parameters, and the right hemidiaphragm was used for the histopathological evaluation. It was observed that the medication administered in group T increased the contraction strength and duration compared with that in group M. Additionally, the duration of semirelaxation was prolonged in group T compared with group M. The highest contraction strength and the longest contraction period among all of the groups were observed in group TM. It was concluded that the combined use of theophylline and methylprednisolone had positive effects on the contraction strength and the durations of contraction and semi-relaxation of the diaphragm muscle. In addition, both drugs had synergistic effects on each other. KEY WORDS: biomechanical parameters; diaphragm muscle; histopathology; methylprednisolone; theophylline.

INTRODUCTION

1

Department of Anesthesiology, Faculty of Medicine, YuzuncuYil University, Van, Turkey 2 Department of Anesthesiology, Faculty of Medicine, Gazi University, Ankara, Turkey 3 Department of Anesthesiology, Faculty of Medicine, İzmir Katip Çelebi University, İzmir, Turkey 4 Department of Biophysics, Faculty of Medicine, Muğla Sıtkı Koçman University, Muğla, Turkey 5 Department of Histology and Embryology, Faculty of Medicine, Sakarya University, Sakarya, Turkey 6 To whom correspondence should be addressed at Department of Anesthesiology, Faculty of Medicine, YuzuncuYil University, Van, Turkey. Email: [email protected]

Respiratory function disorders may vary from mild dysfunction to acute respiratory distress [1], and this impairment may originate at any point along the biological pathway beginning in the central nervous system and terminating in the contractile elements of the peripheral respiratory muscles [2–4]. However, some studies have drawn attention to the possible association of respiratory failure with pathologies of the diaphragm muscle [5, 6]. Fatigue of the respiratory muscles, particularly the diaphragm muscle, impairs alveolar ventilation and gives rise to respiratory failure [7–9].

0360-3997/16/0000-0001/0 # 2016 Springer Science+Business Media New York

Yuzkat, Kati, Isik, Kavak, Goktas, and Cengiz Theophylline commonly used to treat asthma and chronic obstructive pulmonary disease (COPD) through bronchodilator and antiinflammatory effects on the respiratory system. Methylxanthines has also effects on other systems. In addition, theophylline can be used with β2mimetic drugs and corticosteroids in asthma attacks and other clinical conditions, as an initial treatment [10]. Methylxanthines are bronchodilator drugs, the positive effects of which have been shown on the diaphragm contraction in patients with serious respiratory failure [11]. Theophylline increases the contractility of the diaphragm, particularly in the presence of hypoxemia and muscle weakness [12]. However, it is not clear whether theophylline exerts this effect directly on the diaphragm muscle or via other systems because methylxanthines also have systemic effects, such as increasing the secretion of catecholamines, stimulating the respiratory center in the brain, and increasing the strength of the heart beat [13]. In contrast, the long-term and high-dose use of corticosteroids may cause atrophy in the striated muscles due to their negative effects on protein metabolism. The effects of the combined use of two drugs with antagonizing effects on muscle contraction, such as theophylline and methylprednisolone [14, 15] on the biomechanics and histopathology of the diaphragm are not clearly understood. In this study, the effects of the short-term use of theophylline and methylprednisolone were investigated on the biomechanics and histopathology of the normal diaphragm muscle, which are frequently used to treat pathologies of the respiratory system on combined or alone.

MATERIALS AND METHOD The study was approved by the local ethics committee of Yuzuncu Yıl University, according to the International

Helsinki Declaration. The study included 28 male WistarAlbino rats, weighing between 280 and 310 g. Four groups were formed that included seven rats each. The experimental animals were kept in a 12 h light and dark cycle at room temperature conditions. Standard plastic cages were used for shelter and contained three or four animals each. They were fed with ad libitum with standard rat pellets and water. No additional food or antibiotics were used. The room temperature of the animals was kept at 23 ± 1 °C throughout the study. The study included 28 rats; groups were formed that included seven rats each. Four groups made up with rats, theophylline used; named group T, methylprednisolone used; named group M, theophylline plus methylprednisolone used; named group TM, group which was used saline; named group K. In the beginning of the experiment theophylline, 5 mg/kg loading dose was applied to group T and group TM. The medications were continued for 7 days in all of the groups and in two doses administered per day. The rats underwent cervical dislocation under anesthesia on the eighth day, and diaphragm samples were extracted. In associations, the drug dosage schedule is set according to Table 1. Dissection of the diaphragm preparations was entering and had abdominal. For the biomechanical parameters, a muscle stripe was removed from the ventral-costal region of the left hemidiaphragm and placed into a pre-equilibrated (95 % O2 and 5 % CO2) Krebs solution (constituents in mmol/L: 113 NaCl, 4.7 KCl, 1.2 MgSO4, 7 H2O, 1.9 CaCl2, 2 H2O, 1.2 KH2PO4, 25 NaHCO3 11.5 glucose, pH 7.4). For histopathological evaluation, the right hemi-diaphragm was dissected and fixed with 10 % neutral buffered formalin and was embedded into paraffin blocks following routine tissue procedures.

Table 1. Drug Dosage Used in the Experiment Groups

First day

Tn = 7

Morning 5 mg/kg ip theophylline loading dose 2 mg/kg ip metilprednizolon 5 mg/kg ip theophylline loading dose and 2 mg/kg ip metilprednizolon 1 mL ip saline

M n=7 TM n:7 K n=7

ip intraperitoneal

2–7th days

8th day

Evening 1 mg/kg ip theophylline maintenance dose 2 mg/kg ip. metilprednizolon

Morning and evening 1 mg/kg ip theophylline maintenance dose 2 mg/kg ip. metilprednizolon

Medication stopped. Biomechanical and histological evaluation

1 mg/kg ip theophylline maintenance dose and 2 mg/kg İp metilprednizolon 1 mL ip saline

1 mg/kg ip theophylline maintenance dose and 2 mg/kg İp metilprednizolon 1 mL ip saline

Effects Theophylline Methylprednisolone Biomechanical Recordings The study included 28 male rats, weighing between 280 and 310 g. According to the method described above, they were used to prepare the diaphragm. To measure the contraction parameters of the diaphragm muscle, the left diaphragm samples obtained from the rats were properly placed between steel electrodes and suspended over isolated organ baths containing Krebs solution maintained at 37 °C. The bath fluid was continuously exposed to a mixture of 95 % O2 and 5 % CO2 gases. Following the determination of the optimum lengths of the diaphragm muscles in all of the groups, electrical stimuli of 0.5 ms and 15–20 V were given directly with square pulses for 10 min throughout the preparation period via the supra-maximal approach. The diaphragm muscles in all of the groups were primarily stimulated directly with single supra-maximal square pulses, and the isometric tremor contractions were recorded. The tremor contraction strength, the duration of contraction, and the duration of semi-relaxation parameters were determined by a computer program according to the tremor curves. The parameters of contraction and relaxation speeds were also calculated from the curves. Current study biomechanical in register and observation system was used, and Biopac Biopac Systems MP45 data recording and analysis was performed with the program. Histological Analysis The right diaphragm tissues obtained from the rats in all of the groups were embedded into paraffin blocks following routine procedures. Sections of 5 μm thicknesses were obtained from the blocks, and they were deparaffinized for examination under a light microscope (Zeiss Axioskop 40-Germany) to determine the nuclear structures of the tissue, the locations of the nuclei, the serosal structures, cell infiltrations, atrophy, and thickening.

RESULTS The study included 28 rats. The muscle strength values in all of the experimental groups were observed to be higher compared with the control group (p < 0.05). According to control group, it has been found that the contraction strength of group TM is 22 %, the contraction strength of group T is 9 %, and M is 4 % higher. The difference between group T and group M is about 9 % significant (p < 0.05). It has been found that the contraction strength of group TM is higher 9 % than the contraction strength of group T and 18 % than the contraction strength of group M (p < 0.05). The contraction strength was higher in group T compared with group M, and higher in group TM compared with group T and group M (p < 0.05) (Fig. 1 and Table 2). The contraction speed was statistically significantly lower in all of the study groups compared with the control group. No difference was observed between the study groups. The relaxation speed was lower in all of the study groups compared with the control group (p < 0.05). The relaxation speed in group T was lower than those of group TM (35 %) and group M (13 %) (p < 0.05). No difference was observed between group TM and group M. The values obtained for the duration of contraction were greater in all of the groups compared with the control group (p < 0.05). A longer duration of contraction was observed in group TM compared with group T (6.5 %) and group M (11 %), and in group T compared with group M (5.5 %) (p < 0.05). The values obtained for the duration of semi-relaxation were statistically significantly greater in all of the study groups compared with the control group. No difference was observed between the study groups (Table 3). According to the histological evaluation, the nuclear structure was normal and peripherally located in all of the

Statistical Analysis All of the values were expressed as the mean ± SEM (standard error of mean). The number of samples in each group was seven. The consistency of the distribution of the data within the normal range in each group was determined using the Kolmogorov-Smirnov test. Because the number of the factors affecting our variables was three, the univariate multi-factorial ANOVA and two-way ANOVA were used. The level of significance was accepted as p < 0.05. The posthoc Tukey-HSD test was used for the multiple comparisons between the groups. Data analysis was performed with SPSS programa (version10.0) statistical software.

Fig. 1. The contraction strength of the exchange. a, b, and c indicate significant differences in contractile strength between the groups (p < 0.05). a: relative to the control group, b: relative to group M, c: relative to Group T.

Yuzkat, Kati, Isik, Kavak, Goktas, and Cengiz Table 2. Diaphragm Isometric Contractile Characteristics (Mean ± SEM) Groups

Contraction strength (T) (mg)

Contraction speed (+dp/dt) (mg/ms)

Relaxation speed (−dp/dt) (mg/ms)

T M TM K

1250 ± 15.4a,b 1150.5 ± 43.7a 1360.6 ± 50.2a,b,c 1110.3 ± 14.5

8.1 ± 0.42 a 7.8 ± 0.4 a 7.7 ± 0.33 a 8.4 ± 0.6

9.6 ± 0.4a,d 8.5 ± 0.25a 7.1 ± 0.2a 5.6 ± 0.4

SEM standard error of mean Superscript letters indicate significant differences between the groups (p < 0.05): a relative to the control group, b relative to group M, c relative to group T, and d relative to group M and Group TM

groups. The serosal structure was clearly evident significant in all of the groups, whereas no cell infiltration, atrophy, or thickening was observed in any of the groups. No differences were observed between the groups with regard to histological changes.

DISCUSSION New pharmacological applications have recently focused on the enhancement of diaphragmatic contractility and the prevention or reversion of fatigue. In the present study, the normal diaphragm muscle was selected for analysis because it is the most important muscle of the respiratory system. The effects of methylprednisolone, which is believed to have negative effects on the diaphragm muscle in longterm use or high-dose short-term use [16, 17], and theophylline, which has been previously demonstrated to have positive effects on the diaphragm muscle [16–18], were examined. Additionally, the effects of the combined use of methylprednisolone and theophylline on the normal diaphragm muscle were compared with the effects of the individual drugs. There are many factors that influence the function of the diaphragm muscle, including chronic obstructive pulmonary Table 3. Duration of İsometric Contraction of the Diaphragm (Ort. ± SEM) Groups

Duration of contraction (CT) (ms)

Duration of semi-relaxation (1/2RT) (ms)

T M TM K

201.3 ± 9.4&,a 190.4 ± 7.9& 215.2 ± 9.3&,a,b 180.1 ± 5.6

145.6 ± 7.9* 140.8 ± 2.9* 147.6 ± 8.4* 132.5 ± 3.2

SEM standard error of mean Superscript letters and symbols (&,* ) indicate significant differences between the groups (p < 0.05): &relative to the control group, *relative to the control group, a relative to group M, and b relative to group T

disease [16], immobilization, neuromuscular blocker use, sepsis, corticosteroid use [6], long-term mechanical ventilation support [18], aging, shock, and ischemia [5, 19]. In the presence of these factors, the contractility of the diaphragm muscle is negatively affected, and a motion disorder or atrophy is observed. It has been shown in studies that theophylline increases the contraction strength of the diaphragm muscle. However, the mechanism of this increase could not be clearly explained [20, 21]. It has been shown that methylxanthines, such as theophylline and caffeine, at therapeutic doses have provided a 15–50 % improvement in the contraction strength/frequency response curves of the diaphragm muscles in humans and animals in the presence of diaphragmatic fatigue [8]. It has been demonstrated in pharmacotherapeutic applications that prophylactic theophylline administration prevents the weakening of the diaphragm and reverts diaphragmatic fatigue [22, 23]. In an experimental study, the muscle strength was observed to have increased by 1.5 % in the group with of 15 mg/L theophylline added to the isolated organ bath fluid, whereas the increase was 35 % in the group with a 60 mg/L theophylline addition [23]. The work plan of our study has different fiction from the above authors; our aim is to measure the biomechanical parameters to rats by administering drugs intraperitoneal after 1 week. In the present study, the contraction strength in the group with theophylline administration was increased by 13 % compared with the control group. The possible reason for this increase is the inhibition of intracellular calcium release by theophylline and the subsequent stimulation of catecholamine release [23]. It has been demonstrated that glucocorticoids cause a decrease in the contraction strength of the diaphragm muscle depending on the dose and duration of application [24–26]. This decrease in the contraction strength exists via three main properties that are responsible for the formation of strength in the stimulation-contraction mechanism. These properties are the intracellular calcium concentration, the calcium sensitivity of the myofilaments, and the calcium-

Effects Theophylline Methylprednisolone activated maximal strength. Alterations may be observed in the stimulation-contraction mechanism if one or a few of these processes are affected [27]. Sieck et al. [25] administered physiological saline or methylprednisolone to rats for 6 months and observed a 15 % decrease in the isometric muscle strength and maximal muscle strength in the methylprednisolone group. They concluded that the long-term low-dose use of methylprednisolone caused a significant impairment in the functioning of the diaphragm. However, the same effect was not observed following a short-term low-dose corticosteroid treatment. In the present study, the contraction strength observed in the methylprednisolone group was increased by 4 % compared with the control group. We believe that this increase is due to low-dose methylprednisolone use for 7 days. There is limited data in the literature with regard to the effect of the combined use of theophylline and corticosteroids on the respiratory muscles. This type of use resulted in a synergistic effect in the present study. The contraction strength observed in the group with the combined use of theophylline and methylprednisolone was notably increased by 23 % compared with the control group. Furthermore, this increase was higher than that caused by theophylline alone. The contraction strength in the theophylline plus methylprednisolone group exhibited a 9 % increase compared with the theophylline group and an 18 % increase compared with the methylprednisolone group. This may be due to a mechanism whereby theophylline rebalances the impaired calcium homeostasis in the methylprednisolone group, repairs the contractility by normalizing the number of active cross bonds, and increases the contraction strength of the diaphragm. The duration of contraction reflects the Ca+2 released from the sarcoplasmic reticulum, and the duration of hemirelaxation reflects the uptake of Ca+2 from the cytoplasm for storage [27]. In the in vitro study of Golgeli et al. [28] which investigated the effect of theophylline on the duration of contraction, it was observed that theophylline addition to the bath media at a final concentration of 1 millimolar caused no change in the duration of contraction, whereas the semirelaxation period was significantly increased. In our study, the contraction period of the group with theophylline administration was increased by 11 % compared with the control group and by 10 % compared with the methylprednisolone group. The speed of semi-relaxation was increased by 11 % compared with the control group and by 10 % compared with the methylprednisolone group. The increase in the durations of contraction and relaxation may be due to the decreased ion permeability of the cell membrane and decreased activity of specific Ca+2ATPases leading to a

subsequent decrease in the re-uptake of Ca+2 and a delay in the binding of calcium to the filaments. Dekhuijzen et al. [29] used 1 mg/kg triamcinolone daily and 1.25 and 5 mg/kg prednisolone daily in separate groups for 4 weeks and observed severe muscular atrophy of the respiratory and peripheral muscles in the triamcinolone group, whereas no difference was observed in the control group or the two prednisolone groups. Furthermore, no significant difference was observed in the contraction or semi-relaxation periods of the prednisolone group regarding the contraction properties of the diaphragm [25]. In the study of Fletcher et al. [30], 5 mg/kg/daily prednisolone was administered for 8 days, and the semi-relaxation period was observed to be significantly increased. In the present study, according to the diaphragm contraction curve, the contraction period in the methylprednisolone group was increased by 7 %, and the semi-relaxation period was increased by 9 % compared with the control group. In the group with the combined use of theophylline and methylprednisolone, the contraction period was increased by 21 %, and the semi-relaxation period was increased by 14 % compared with the control group. The contraction strength and the contraction period were significantly increased in this group. According to these data, we believe that theophylline and methylprednisolone have synergistic effects on the diaphragm. Viires et al.[18] administered theophylline and methylprednisolone to rats that were mechanically ventilated and examined the diaphragm muscle in which they observed a decrease in the membranous diameters caused by the mechanical ventilation. In contrast, theophylline administration caused an increase in the membranous diameters. Corticosteroids may cause myopathy, both in the skeletal muscles and in the respiratory muscles. This is an important clinical situation, particularly for patients with chronic obstructive pulmonary disease, which is characterized by the loss of function in the respiratory muscles. Short-term high-dose corticosteroid use results in diffuse muscular weakness, acute myopathy characterized by severe dyspnea, rhabdomyolysis, and atrophy [17, 31]. The pathophysiology of the myopathy caused by steroids is unknown. However, it may be explained by the effects of steroids on the reduction of protein synthesis and the increase in glycogen accumulation. Decreases in body mass and diaphragm mass have been demonstrated following corticosteroid administration in animal models [17]. Ferguson et al. [32] investigated the pathological changes that were experimentally induced in the respiratory muscles by the use of corticosteroids and determined that the microscopic changes were mostly observed in the diaphragm muscle. Dekhuijzen et al. [29]

Yuzkat, Kati, Isik, Kavak, Goktas, and Cengiz reported that 1.25 mg/kg/day of prednisolone did not cause a significant histological change in the diaphragm muscle, but 5 mg/kg/day prednisolone and triamcinolone treatment resulted in significant atrophy. In the study by Eason et al. [33] on male rats, the atrophic effect of dexamethasone on the diaphragm muscle was observed to be 14 %. In the same study, no difference was observed between the groups regarding the nuclear structure, the location of the nuclei, the serosal structure, cell infiltration, atrophy, or thickening. In our study, the steroid was as administered at a dose of 2 mg/kg/day, and no steroid-related diaphragmatic atrophy such as that observed in the studies with short-term, low-dose steroid administration was observed. Weakness of this study, we used an experimental model which has not been performed on the hypoxic or tired diaphragm muscle. Also tetanic stimulus which will perform a diaphragm fatigue has not been given. However, patients with chronic illness and critically ill which tires diaphragm would fine better its.

CONCLUSION Treatments with theophylline alone or methylprednisolone alone were found to have positive effects on the physiology of the diaphragm muscle; however, the highest values were obtained in the group treated with the combined use of these two agents. We also believe that methylprednisolone and theophylline combined have synergistic effects on the contraction strength of the diaphragm muscle and on the durations of this contraction and semirelaxation by potentiating the effects of each other. ACKNOWLEDGMENTS This study was supported by Yuzuncu Yil University Research Foundation (2011-TFF-U 025).

COMPLIANCE WITH ETHICAL STANDARDS Conflict of Interest. The authors declare that they have no conflict of interest.

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